Non-anaphylactogenic IgE vaccines

ABSTRACT

The present invention provides compositions and methods for the use of antigenic peptides derived from the Fc portion of the epsilon heavy chain of IgE molecules from two unrelated species as vaccines for the treatment and prevention of IgE-mediated allergic disorders. In particular, the invention provides compositions for the treatment and prevention of IgE-mediated allergic disorders comprising an immunogenic amount of one or more antigenic peptides.

FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods for theuse of antigenic peptides derived from the Fc portion of the epsilonheavy chain of IgE molecules as vaccines for the treatment andprevention of IgE-mediated allergic disorders. In particular, thepresent invention relates to compositions comprising at least oneantigenic peptide comprising an amino acid sequence derived from the CH3domain of IgE molecules from two different species for the treatment orprevention of an IgE-mediated allergic disorder. The present inventionalso relates to compositions comprising antigenic peptides coupled toheterologous carrier proteins and optionally further comprising anadjuvant. The compositions of the present invention induce anti-IgEantibodies which bind to soluble (free) IgE in serum and other bodilyfluids, prevent IgE from binding to its high affinity receptors on mastcells and basophils, and do not cross-link receptor-bound IgE. Thepresent invention further relates to methods of administeringcompositions of the invention to animals, preferably mammals and mostpreferably humans, for the treatment or prevention of IgE-mediatedallergic disorders.

BACKGROUND OF THE INVENTION

[0002] Immune-mediated allergic (hypersensitivity) reactions areclassified into four types (I-IV) according to the underlying mechanismsleading to the expression of the allergic symptoms. Type I allergicreactions are characterized by IgE-mediated release of vasoactivesubstances such as histamine from mast cells and basophils. The releaseof these substances and the subsequent manifestation of allergicsymptoms are initiated by the cross-linking of allergen-bound IgE to itsreceptor on the surface of mast cells and basophils.

[0003] An IgE antibody is a complex molecule consisting of two identicalheavy chains and two identical light chains held together by disulfidebonds in a “Y” shape-configuration. Each light chain consists of avariable (V_(L)) domain linked to a constant domain (C_(L)), and eachheavy chain consists of a variable domain (V_(H)) and four constantdomains (CH1, CH2, CH3, and CH4, also known as Cε1, Cε2, Cε3, and Cε4;respectively). The two arms of an IgE antibody contain the site at whichan IgE antibody binds to its specific antigen (allergen) and each arm isreferred to as a Fab (fragment-antigen-binding) fragment. The tail of anIgE antibody is termed Fc (fragment-crystalline) as it can form crystalswhen separated from the Fab fragments of the antibody under appropriateexperimental conditions. The Fc fragment of an IgE antibody consists ofthe CH2, CH3, and CH4 domains and contains the biologically activestructures of the IgE antibody (e.g., receptor binding sites).

[0004] The production of IgE antibodies requires interactions andcollaborations among three cells; antigen presenting cells (APC), Tlymphocytes (T helper cells; Th) and antibody producing cells (Blymphocytes; B cells). When a foreign substance, an allergen, isintroduced for the first time into the body of subjects (e.g., byinhalation of environmental allergen, ingestion of certain foods, or viathe skin), the allergen is taken up by APC's (e.g., macrophages) whichthen digest or process the allergen into smaller fragments (epitopes).These fragments are displayed on the surface of APC's in associationwith specific molecules known as major histocompatibility complexproteins. The allergen fragment/MHC complex displayed on the surface ofAPC's is recognized and bound by receptors on the surface of specific Tlymphocytes. This recognition and binding event leads to the activationof T lymphocytes and the subsequent expression and secretion ofcytokines such as interleukin-4 (IL-4). These cytokines induce themultiplication, clonal expansion and differentiation of B cells specificfor the allergen in question (i.e., B cell which express on theirsurface immunoglobulin receptors capable of binding to the allergen) andultimately lead to the production of IgE antibodies from these B cells.A portion of the activated T lymphocytes and IgE producing B cellseventually become committed to a pool of cells called T and B memorycells, which are capable of faster recognition of allergen uponsubsequent exposure to the allergen.

[0005] In individuals suffering from type I allergic reactions, exposureto an allergen for a second time leads to the production of high levelsof IgE antibodies specific for the allergen as a result of theinvolvement of memory B and T cells in the 3-cell interaction requiredfor IgE production. The high levels of IgE antibodies produced cause anincrease in the cross-linking of IgE receptors on mast cells andbasophils by allergen-bound IgE, which in turn leads to the activationof these cells and the release of the pharmacological mediators that areresponsible for the clinical manifestations of type I allergic diseases.

[0006] Two receptors with differing affinities for IgE have beenidentified and characterized. The high affinity receptor (FcεRI) isexpressed on the surface of mast cells and basophils. The low affinityreceptor (FcεRII/CD23) is expressed on many cell types including Bcells, T cells, macrophages, eosinophils and Langerhan cells. The highaffinity IgE receptor consists of three subunits (alpha, beta and gammachains). Several studies demonstrate that only the alpha chain isinvolved in the binding of IgE, whereas the beta and gamma chains (whichare either transmembrane or cytoplasmic proteins) are required forsignal transduction events. The identification of IgE structuresrequired for IgE to bind to the FcεRI on mast cells and basophils is ofutmost importance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

[0007] Over the last 15 years, a variety of approaches have beenutilized to determine the FcεRI binding site on IgE. These approachescan be classified into five different categories. In one approach, smallpeptides corresponding to portions of the Fc part of an IgE moleculewere produced and analyzed for their ability to inhibit IgE from itsreceptors. See, for example, Nakamura et al., EP0263655 published Apr.13, 1988, Burt et al., 1987, European Journal of Immunol., 17:437-440;Helm et al., 1988, Nature 331:180-183; Helm et al., 1989, PNAS86:9465-9469; Vercelli et al., 1989, Nature 338:649-651; Nio et al.,1990, Peptide Chemistry, 2: 203-208; Nio et al., 1993, FEBS Lett.319:225-228; and Nio et al., 1992, FEBS Lett. 314:229-231. Although manyof the peptides described in these studies were shown to inhibit thebinding of IgE to its receptors, different studies reported differentsequences as being responsible for IgE binding.

[0008] Helm et al. (1988, Nature 331:180-183) identified a 75 amino acidpeptide that spans the junction between CH2 and CH3 domains of IgE andshowed that this peptide binds to the IgE receptor with an affinityclose to that of the native IgE molecule. On the other hand, Basu et al.(1993, Journal of Biological Chemistry 268: 13118-13127) expressedvarious fragments from IgE molecules and found that only those fragmentscontaining both the CH3 and CH4 domains were able to bind IgE and thatCH2 domain is not necessary for binding. Vangelista et al. (1999,Journal of Clinical Investigation 103:1571-1578) expressed only the CH3domain of IgE and showed that this domain alone could bind to IgEreceptor and prevent binding of IgE to its receptor. The results of Basuet al. and Vangelista et al. are inconsistent and conflict with those ofHelm et al. cited above.

[0009] In a second approach to identify the FcεRI binding site on IgE,polyclonal antibodies against peptides corresponding to parts of the CH2domain, CH3 domain or CH4 domain were produced and used to probe forreceptor binding site on IgE (Robertson et al., 1988, Molecular Immunol.25:103-118). Robertson et al. concluded that the amino acid residuesdefined by a peptide derived from the CH4 domain were not likely to beinvolved in receptor binding, whereas amino acid residues defined by apeptide derived from the CH3 domain of IgE were most likely proximal tothe IgE receptor-binding site (amino acids 387-401). However, theanti-CH3 peptide antibodies induced in response to the CH3 peptidereleased histamine from IgE-loaded mast cells indicating that the aminoacids defined by the CH3 peptide did not define the bona fide IgEreceptor-binding site and that anti-CH3 peptide antibodies could causeanaphylaxis.

[0010] In a third approach to identify the FcεRI binding site on IgE,several investigators produced IgE mutants in an attempt to identify theamino acid residues involved in receptor binding (see, e.g., Schwarzbaumet al., 1989, European Journal of Immunology 19:1015-1023; Weetall etal., 1990, Journal of Immunology 145:3849-3854; and Presta et al., 1994,Journal of Biological Chemistry 269:26368-26373). Schwartzbaum et al.demonstrated that an IgE antibody with the point mutation proline tohistidine at amino acid residue 442 in the CH4 domain has a two foldreduced affinity for the IgE receptor. Schwartzbaum et al. concludedthat the CH4 domain of an IgE antibody is involved in IgE binding to itsreceptor. However, Schwartzbaum's conclusion contradict Weetall et al.'sconclusion that the binding of IgE to its high affinity receptorinvolves portions of the CH2 and CH3 domains of the IgE antibody, butnot the CH4 domain. Further, Schwartzbaum et al.'s conclusionscontradict Presta et al.'s conclusion that the amino acid residues ofthe IgE antibody important for binding to the FcεRI are located in theCH3 domain.

[0011] In a fourth approach to identify the FcεRI binding site on IgE,chimeric IgE molecules were constructed and analyzed for their abilityto bind to the FcεRI. Weetall et al., supra constructed a series ofchimeric murine IgE-human IgG molecules and tested their binding to theIgE receptor. Weetall et al., supra concluded that the CH4 domain doesnot participate in receptor binding and that the CH2 and CH3 domains areboth required for binding to the high affinity receptor on mast cells.In another study, Nissim et al. (1993, Journal of Immunol 150:1365-1374)tested the ability of a series of human IgE-murine IgE chimera to bindto the FcεRI and concluded that only the CH3 domain is needed forbinding to the FcεRI. The conclusion by Nissim et al. corroborates theconclusion by Vangelista et al. that the CH3 domain of IgE alone bindsto the FcεRI. However, the conclusions by Nissim et al. and Vangelistaet al. contradict the conclusions of Weetall et al. and Robertson et al.

[0012] Presta et al., supra produced chimeric human IgG in which theCγH2 was replaced with CH3 from human IgE. When tested for receptorbinding, this chimera bound to the FcεRI albeit with a four-fold reducedaffinity compared with native IgE. The results of Presta et al. appearto corroborate with the results of Nissim et al., but conflict withthose of Weetall et al., Helm et al., and Basu et. al., cited above. Ina further attempt to define the exact amino acid residues responsiblefor the binding of IgE to its receptor, Presta et al. inserted specificamino acid residues corresponding to CH2-CH3 hinge region and threeloops from the CH3 domain of human IgE into their analogous locationswithin human IgG and called these mutants IgGEL. Unfortunately, whenthese IgGEL variants were tested for receptor binding, they exhibitedminimal binding compared to the native IgE or the IgG in which the fulllength IgE CH3 domain replaced the full length CγH2 domain. In a fifthapproach to identify the FcεRI binding site on IgE, monoclonalantibodies have been developed and analyzed for their ability to blockIgE binding to the FcεRI. See, for example, Del Prado et al., 1991,Molecular Immunology 28:839-844; Keegan et al., 1991, MolecularImmunology 28:1149-1154; Hook et al., 1991, Molecular Immunology28:631-639; Takemoto et al., 1994, Microbiology and Immunology 38:63-71;and Baniyash et al., 1988, Molecular Immunology 25:705-711. Althoughmany monoclonal antibodies have been developed, they have providedlittle information on the bona fide IgE receptor-binding site because inmany cases the amino acid sequence recognized by these monoclonalantibodies have not or could not be identified. Further, the monoclonalantibodies developed may block IgE from binding to its receptor bysteric hindrance or induction of severe conformational changes in theIgE molecule, rather than by the binding and masking of IgE residuesdirectly involved in receptor binding.

[0013] It is apparent from the above discussion that approaches thathave been devised to identify the receptor binding site on IgE haveproduced conflicting results. The difficulty in the identification ofthe amino acid residues of IgE responsible for receptor binding could befurther complicated by the possibility that the site on IgE used forbinding to the receptor may not be a linear sequence of amino acids,which could be mimicked by a synthetic peptide. Rather, the binding sitemay be a conformational determinant formed by multiple amino acids thatare far apart in the IgE protein sequence which are brought into closeproximity only in the native three-dimensional structure of IgE. Studieswith IgE variants, IgE chimera, and monoclonal anti-IgE antibodiesstrongly suggest that the binding site is a conformational determinant.

[0014] Currently, IgE-mediated allergic reactions are treated with drugssuch as antihistamines and corticosteroids which attempt to alleviatethe symptoms associated with allergic reactions by counteracting theeffects of the vasoactive substances released from mast cells andbasophils. High doses of antihistamines and corticosteroids havedeleterious side effects such as renal and gastrointestinal toicities.Thus, other methods for treating type I allergic reactions are needed.

[0015] One approach to the treatment of type I allergic disorders hasbeen the production of monoclonal antibodies which react with soluble(free) IgE in serum, block IgE from binding to its receptor on mastcells and basophils, and do not bind to receptor-bound IgE (i.e., theyare non-anaphylactogenic). Two such monoclonal antibodies (rhuMab E25and CGP56901) are in advanced stages of clinical development fortreatment of IgE-mediated allergic reactions (see, e.g., Chang, T. W.,2000, Nature Biotechnology 18:157-62). The identity of the amino acidresidues of the IgE molecule recognized by these monoclonal antibodiesare not known and it is presumed that these monoclonal antibodiesrecognize conformational determinants on IgE.

[0016] Although early results from clinical trials with therapeuticanti-IgE monoclonal antibodies suggest that these therapies arceffective in the treatment of atopic allergies, the use of monoclonalantibodies for long-term treatment of allergies has some significantshortcomings. First, since these monoclonal antibodies were originallyproduced in mice, they had to be reengineered so as to replace mousesequences with consensus human IgG sequences (Presta et al., 1993, TheJournal of Immunology 151:2623-2632). Although this “humanization”process has led to production of monoclonal antibodies that contain 95%human sequences, there remain some sequences of mouse origin. Sincetherapy with these anti-IgE antibodies requires frequent administrationof the antibodies over a long period of time, some treated allergicpatients could produce an antibody response against the mouse sequencesthat still remain within these therapeutic antibodies. The induction ofantibodies against the therapeutic anti-IgE would negate the therapeuticimpact of these anti-IgE antibodies at least in some patients. Second,the cost of treatment with these antibodies will be very high since highdoses of these monoclonal antibodies are required to induce atherapeutic effect. Moreover, the frequency and administration routeswith which these antibodies have to be administered are inconvenient. Amore attractive strategy for the treatment of IgE-mediated disorders isthe administration of peptides which induce the production of anti-IgEantibodies.

[0017] One of the most promising treatments for IgE-mediated allergicreactions is the active immunization against appropriatenon-anaphylactogenic epitopes on endogenous IgE. Stanworth et al. (U.S.Pat. No. 5,601,821) described a strategy involving the use of a peptidederived from the CH4 domain of the human IgE coupled to a heterologouscarrier protein as an allergy vaccine. However, this peptide has beenshown not to induce the production of antibodies that react with nativesoluble IgE. Further, Hellman (U.S. Pat. No. 5,653,980) proposedanti-IgE vaccine compositions based on fusion of full length CH2-CH3domains (approximately 220 amino acid long) to a foreign carrierprotein. However, the antibodies induced by the anti-IgE vaccinecompositions proposed in Hellman will most likely result in anaphylaxissince antibodies against some portions of the CH2 and CH3 domains of theIgE molecule have been shown to cross-link the IgE receptor on thesurface of mast cell and basophils and lead to production of mediatorsof anaphylaxis (see, e.g., Stadler et al., 1993, Int. Arch. Allergy andImmunology 102:121-126). Therefore, a need remains for vaccines for thetreatment of IgE-mediated allergic reactions which do not induceanaphylactic antibodies.

[0018] The significant concern over induction of anaphylaxis hasresulted in the development of another approach to the treatment of typeI allergic disorders consisting of mimotopes that could induce theproduction of anti-IgE polyclonal antibodies when administered toanimals (see, e.g., Rudolf, et al., 1998, Journal of Immunology160:3315-3321). Kricek et al. (International Publication No. WO97/31948) screened phage-displayed peptide libraries with the monoclonalantibody BSW17 to identify peptide mimotopes that could mimic theconformation of the IgE receptor binding. These mimotopes couldpresumably be used to induce polyclonal antibodies that react with freenative IgE, but not with receptor-bound IgE as well as block IgE frombinding to its receptor. Kricek et al. disclosed peptide mimotopes thatare not homologous to any part of the IgE molecule and are thusdifferent from peptides disclosed in the present invention.

[0019] A major obstacle facing the development of an anti-IgE vaccine isthe lack of information regarding the precise amino acids representingnon-anaphylactogenic IgE determinants that could be safely used toimmunize allergic subjects and induce non-anaphylactogenic polyclonalantibodies (i.e., polyclonal anti-IgE antibodies that do not bind toreceptor-bound IgE). The peptide compositions of the present inventionare selected to be non-anaphylactogenic; i.e., the peptide compositionsdo not result in production of anti-IgE antibodies that could bind orcause cross-linking of IgE bound to mast cells or basophils. Thuspeptides of the present invention have superior safety profile and aredifferentiated by sequence composition from disclosed vaccines based onfull-length C2H-CH3 domains.

SUMMARY OF THE INVENTION

[0020] The present invention provides compositions and methods for theuse of antigenic peptides derived from the Fc portion of the epsilonheavy chain of IgE molecules as vaccines for the treatment andprevention of IgE-mediated allergic disorders. In one embodiment, theinvention provides compositions for the treatment and prevention ofIgE-mediated allergic disorders comprising an immunogenic amount of oneor more antigenic peptides derived from the CH3 domains of IgE moleculesfrom two unrelated species effective for treatment or prevention of anIgE-mediated allergic disorder. Preferably, compositions of the presentinvention comprise an immunogenic amount of one or more antigenicpeptides comprising the amino acid sequence of SEQ ID NOS: 2, 3, 10, 11,12, 13 or 14 or an antigenic fragment, derivative or variant thereof.

[0021] The antigenic peptides can be supplied by direct administrationor indirectly as “pro-drugs” using somatic cell gene therapy.

[0022] In a preferred embodiment, the present invention is based, inpart, on the discovery that antigenic peptides comprising conservedamino acid residues of the CH3 domain of an IgE molecule from onespecies flanked by variable amino acid residues of the CH3 domain of anIgE molecule from a second unrelated species are capable of inducing ahigh titer of anti-IgE antibodies when administered to an animal withoutcausing anaphylaxis. The Applicants compared the primary amino acidsequences of IgE molecules from different species, e.g., rat IgE and dogIgE, and identified conserved amino acid residues in the CH3 domains ofthe IgE molecules from the different species. The Applicants alsodetermined that the conserved amino acid residues in the CH3 domains ofIgE molecules from different species are flanked by amino acid residuesthat vary from species to species (referred to as “the variable aminoacid residues”).

[0023] Accordingly, in one embodiment, the present invention encompassesantigenic peptides comprising amino acid residues of the CH3 domain ofan IgE molecule from a first species flanked by amino acid residues ofthe CH3 domain of an IgE molecule from a second unrelated species. Theamino acid residues of the CH3 domain of the IgE molecule from the firstspecies, which comprise the antigenic peptide, are conserved in the CH3domain of the IgE molecule of the second unrelated species. However, theamino acid residues of the CH3 domain of the IgE molecule of the secondunrelated species which comprise the antigenic peptide are not conserved(i.e., vary) in the CH3 domain of the IgE molecule of the first species.Thus, for example, an antigenic peptide of the present invention couldcomprise conserved amino acid residues of the CH3 domain of the canineIgE molecule flanked by amino acid residues of the CH3 domain of the ratIgE molecule. Such an antigenic peptide would preferably be administeredto a dog to treat or prevent an IgE-mediated allergic disorder. Thepresent invention further provides antigenic fusion proteins derivedfrom a single species, which do not cause anaphylaxis when administeredto an animal. Preferably, such an antigenic fusion protein having thesequence SEQ ID NO: 27.

[0024] The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3 domains of IgE molecules from the same orfrom two or more unrelated species and one or more pharmaceuticallyacceptable carriers. In one embodiment, a pharmaceutical composition ofthe invention comprises an immunogenically effective amount of one ormore antigenic peptides derived from the CH3 domains of IgE moleculesfrom two unrelated species and one or more pharmaceutically acceptablecarriers. In another embodiment, a pharmaceutical composition of theinvention comprises one or more pharmaceutical carriers and animmunogenically effective amount of one or more antigenic peptidederived from the CH3 domains of IgE molecules from two unrelated species(SEQ ID NOS: 2, 3, and 10-14) and a heterologous carrier protein such asSEQ ID NOS: 9 and 23.

[0025] The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3 domains of IgE molecules from twounrelated species, a pharmaceutically acceptable carrier, and anadjuvant. Adjuvants encompass any compound capable of enhancing animmune response to an antigen. Examples of adjuvants which may beeffective, include, but are not limited to: aluminum hydroxide,monophosphoryl lipid A (MPLA) -acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,simple immunostimulatory oligonucleotides, cytokines such as IL-12, IL-2or IL-1, saponins, and microbial toxins such as cholera toxin, heatlabile toxin and genetically altered derivatives of them.

[0026] In another embodiment, a pharmaceutical composition of theinvention comprises a pharmaceutical carrier, an adjuvant and animmunogenically effective amount of one or more antigenic fusionproteins comprising an antigenic peptide derived from the CH3 domains ofIgE molecules from two unrelated species and a heterologous carrierprotein. In a preferred embodiment, a pharmaceutical composition of theinvention comprises a pharmaceutical carrier, an adjuvant and animmunogenically effective amount of one or more antigenic peptidescomprising of the amino acid sequence of SEQ ID NOS: 2, 3 and 10-14.

[0027] In another preferred embodiment, a pharmaceutical composition ofthe present invention comprises a pharmaceutical carrier, an adjuvant,and an immunogenically effective amount of one or more fusion proteinscomprising the amino acid sequence of SEQ ID NOS: 2, 3, and 10 to 14.

[0028] The present invention also provides methods of administeringcompositions of the invention to animals, preferably mammals and mostpreferably humans for the treatment or prevention of IgE-mediatedallergic disorders. The compositions of the present invention are insuitable formulation to be administered to animals, preferably mammalssuch as companion animals (e.g., dogs, cats, and horses) and livestock(e.g., cows and pigs), and most preferably humans. The compositions ofthe invention are administered in an amount effective to elicit animmune response, for example, the production of polyclonal antibodieswith specificity for an IgE molecule. In one embodiment, thecompositions of the invention are administered in an amount effective toinduce the production of polyclonal or monoclonal antibodies withspecificity for the Fc portion of an IgE molecule required for IgE tobind to its receptor (i.e., the CH3 domain of an IgE molecule). In apreferred embodiment, the compositions of present invention areadministered in an amount effective to induce the production of anti-IgEantibodies which bind to soluble (free) IgE in serum and other bodilyfluids, prevent IgE from binding to its high affinity receptors on mastcells and basophils, and do not cross-link receptor-bound IgE.Accordingly, the compositions of the invention are administered in anamount effective to induce the production of polyclonal antibodies whichdo not induce anaphylaxis for the treatment or prevention ofIgE-mediated allergic disorders.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1. Baculovirus expressed human CH3 domain separated bySDS-SAGE on 4-12% gels under reducing conditions. The 11 kDA CH3 domaincan be seen in lane 4. No corresponding bands were observed in the sf-9cell control (lane 2) or in wild type baculovirus (lane 3). Positions ofmolecular mass standards (kDa) are indicated in lane 1.

[0030]FIG. 2. Immunoblotting of baculovirus expressed Human CH3 domainwith rabbit A# 145 RBS-2 antiserum. Samples were separated by SDS-SAGEon 4-12% gels under reducing conditions. The 11 kDA CH3 domain can beseen in lane 4. No bands were observed in the sf-9 cell control (lane 2)or in wild type baculovirus (lane 3). Positions of molecular massstandards (kDa) are indicated in lane 1.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides compositions and methods for theuse of antigenic peptides derived from the Fc portion of the epsilonheavy chain of IgE molecules as vaccines for the treatment andprevention of IgE-mediated allergic disorders. In particular, thepresent invention provides compositions comprising an immunogenic amountof an antigenic peptide derived from the CH3 domains of IgE moleculesfrom two unrelated species effective for treatment or prevention of anIgE-mediated allergic disorder. Preferably, compositions of the presentinvention comprise an immunogenic amount of one or more antigenicpeptides comprising the amino acid sequence of SEQ ID NOS: 1 to 6 and 10to 14.

[0032] The antigenic peptides of the present invention comprise an aminoacid sequence of the CH3 domains of IgE molecules from two unrelatedspecies and induce the production of anti-IgE antibodies, which are notanaphylactic. In particular, the antigenic peptides of the presentinvention induce the production of anti-IgE antibodies which bind tosoluble (free) IgE in serum and other bodily fluids, prevent IgE frombinding to its high affinity receptors on mast cells and basophils, anddo not cross-link receptor-bound IgE. The antigenic peptides of thepresent invention may be coupled to one or more heterologous peptides.The antigenic peptides of the invention can be supplied by directadministration or indirectly as “pro-drugs” using somatic cell genetherapy.

[0033] In one embodiment, an antigenic peptide of the inventioncomprises an amino acid sequence comprising amino acid residues of theCH3 domain of an IgE molecule from a first species flanked by amino acidresidues of the CH3 domain of an IgE molecule from a second, preferablyunrelated, species. An antigenic peptide of the invention comprises atleast 10 amino acid residues of the CH3 domain of an IgE molecule from afirst species, at least 15 amino acid residues of the CH3 domain of anIgE molecule from a first species, at least 20 amino acid residues ofthe CH3 domain of an IgE molecule from a first species, or at least 25amino acid residues of the CH3 domain of an IgE molecule from a firstspecies. Further, an antigenic peptide of the invention comprises atleast 10 amino acid residues of the CH3 domain of an IgE molecule from asecond species, at least 15 amino acid residues of the CH3 domain of anIgE molecule from a second species, at least 20 amino acid residues ofthe CH3 domain of an IgE molecule from a second species, or at least 25amino acid residues of the CH3 domain of an IgE molecule from a secondspecies.

[0034] In specific embodiments, an antigenic peptide of the invention isat least 10 amino acid residues long, at least 15 amino acid residueslong, at least 20 amino acid residues long, or at least 25 amino acidresidue long, or at least 30 amino acid residues long. In a preferredembodiment, an antigenic peptide of the invention comprises an aminoacid sequence comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second unrelated species and saidantigenic peptide is between 28 and 31 amino acid residues. The presentinvention also provides antigenic fusion proteins comprising anantigenic peptide and a heterologous carrier protein. In a specificembodiment, an antigenic fusion protein comprises amino acid residues ofthe CH3 domain of an IgE molecule from a first species flanked by aminoacid residues of the CH3 domain of an IgE molecule from a secondunrelated species and a heterologous protein carrier. In a preferredembodiment, an antigenic fusion protein of the present inventioncomprises the amino acid sequence of SEQ ID NOS: 2, 3, and 10 to 14.

[0035] The present invention also provides antigenic peptides orantigenic fusion proteins of the invention in which one or more aminoacid substitutions, additions or deletions has been introduced.Mutations can be introduced by standard techniques known to those ofskill in the art. For example, one or more mutations at the nucleotidelevel which result in one or more amino acid mutations can be introducedby site-directed mutagenesis or PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened fortheir ability to induce anti-IgE antibodies which do not causeanaphylaxis.

[0036] The present invention also provides methods for treating orpreventing IgE-mediated allergic disorders in animals, preferablymammals and most preferably humans, comprising administeringpharmaceutical compositions, which do not induce anaphylaxis. Thepharmaceutical compositions to be administered in accordance with themethods of the present invention encompass antigenic peptides derivedfrom the CH3 domains of IgE molecules from two unrelated species. Thepharmaceutical compositions to be administered in accordance with themethods of the present invention also include: (i) recombinant antigenicpeptides having an amino acid sequence comprising amino acid residues ofthe CH3 domain of an IgE molecule from a first species flanked by aminoacid residues of the CH3 domain of an IgE molecule from a secondspecies; (ii) recombinant antigenic fusion proteins comprising aminoacid residues of the CH3 domain of an IgE molecule from a first speciesflanked by amino acid residues of the CH3 domain of an IgE molecule froma second species and a heterologous carrier protein; (iii) plasmidcompositions comprising polynucleotide encoding an antigenic peptidehaving an amino acid sequence comprising amino acid residues of the CH3domain of an IgE molecule from a first species flanked by amino acidresidues of the CH3 domain of an IgE molecule from a second species; and(iv) plasmid compositions comprising polynucleotides encoding forantigenic fusion proteins comprising amino acid residues of the CH3domain of an IgE molecule from a first species flanked by amino acidresidues of the CH3 domain of an IgE molecule from a second species anda heterologous carrier protein.

[0037] In one embodiment, a pharmaceutical composition of the presentinvention comprises one or more antigenic peptides having the amino acidsequence comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second species. In a preferredembodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic peptides between 28 and 31 amino acidresidues long having the amino acid sequence comprising amino acidresidues of the CH3 domain of an IgE molecule from a first speciesflanked by amino acid residues of the CH3 domain of an IgE molecule froma second unrelated species. In accordance with these embodiments, thepharmaceutical compositions may further comprise an adjuvant.

[0038] The present invention also provides pharmaceutical compositionscomprising one or more antigenic fusion proteins. In a specificembodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic fusion proteins comprising an antigenicpeptide of the invention and a heterologous carrier protein. Inaccordance with this embodiment, the pharmaceutical composition mayfurther comprise an adjuvant.

[0039] As used herein the term “heterologous carrier protein” refers toa protein which does not possess high homology to a protein found in thespecies that is receiving a composition of the invention and elicits animmune response. A protein possesses high homology if it is greater thanat least 75% identical, more preferably at least 85% identical or atleast 90% identical to a protein as determined by any known mathematicalalgorithm utilized for the comparison of two amino acid sequences (see,e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877; Torellis and Robotti, 1994, Comput. Appl. Biosci. 10: 3-5;and Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. 85: 2444-8).Preferably, the percent identity of two amino acid sequences isdetermined by BLAST protein searches with the XBLAST program, score=50,wordlength=3. Examples of heterologous carrier proteins include, but arenot limited to, SEQ ID NOS: 7, 8 or 9, KLH, PhoE, and rmLT.

[0040] A heterologous carrier protein can be fused to the N-terminus,C-terminus or both termini of an antigenic peptide of the invention.Antigenic fusion proteins of the invention can be produced by techniquesknown to those of skill in the art, for example, by standard recombinantDNA techniques. For example, a nucleotide sequence encoding an antigenicfusion protein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a gene sequenceencoding an antigenic fusion protein (see, e.g., Ausubel et al., infra).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A nucleic acidencoding an antigenic peptide of the invention can be cloned into suchan expression vector such that the fusion moiety is linked in-frame tothe antigenic peptide of the invention.

[0041] In a specific embodiment, a pharmaceutical composition of thepresent invention comprises an antigenic peptide having an amino acidsequence comprising amino acid residues of SEQ ID NOS 2, 3 and 10 to 14.

[0042] In another embodiment, a pharmaceutical composition of thepresent invention comprises an antigenic fusion protein comprising theamino acid sequence of SEQ ID NOS: 2, 3 and 10 to 14. In accordance withthese embodiments, the pharmaceutical compositions may further comprisean adjuvant.

[0043] The pharmaceutical compositions of the present invention are insuitable formulation to be administered to animals such as companionanimals (e.g., dogs and cats) and livestock (e.g., pigs, cows andhorses) and humans for the treatment or prevention of IgE-mediatedallergic disorders.

[0044] IgE mediated disorders include allergic rhinitis/hay fever,asthma, atopic dermatitis, flea allergy, food allergy and inhalantallergy.

[0045] Preferably, a pharmaceutical composition of the inventioncomprising an antigenic peptide of the invention is administered to thesame species as the amino acid residues derived from the CH3 domain ofan IgE molecule of the first species to treat or prevent an IgE-mediatedallergic disorder. IgE-mediated allergic disorders include, but are notlimited to, asthma, allergic rhinitis, gastrointestinal allergies suchas food allergies, eosinophilia, and conjunctivitis. The pharmaceuticalcompositions of the invention are administered to a subject (an animal)in an amount effective for the treatment, prevention or inhibition ofIgE-mediated allergic disorders, or an amount effective for inducing ananti-IgE response that is not anaphylactic, or an amount effective forinhibiting or reducing the release of vasoactive substances such ashistamine, or an amount effective for alleviating one or more symptomsassociated with an IgE-mediated allergic disorder.

[0046] The pharmaceutical compositions of the invention can be used withany known method of treating IgE-mediated allergic disorders. In oneembodiment, one or more pharmaceutical compositions of the invention andone or more antihistamines are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In anotherembodiment, one or more pharmaceutical compositions of the invention andone or more corticosteroids are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In yetanother embodiment, one or more pharmaceutical compositions of theinvention and one or more anti-IgE monoclonal antibodies (e.g., BSW17)are administered to an animal for the treatment or prevention of anIgE-mediated allergic disorder.

[0047] The present invention encompasses polynucleotide sequencesencoding the antigenic peptides (SEQ ID NOS: 2, 3 and 10 to 14), carrierproteins (SEQ ID NOS: 7, 8 and 9) or antigenic fusion proteins (SEQ IDNOS: 2, 3, and 10 to 14) of the invention. The present inventionprovides nucleic acid molecules comprising different polynucleotidesequences due to the degeneracy of the genetic code which encodeidentical antigenic peptides and antigenic fusion proteins. Thepolynucleotide sequence of a CH3 domain of an IgE molecule can beobtained from scientific literature, Genbank, or using cloningtechniques known to those of skill in the art. In particular, thepresent invention encompasses polynucleotide sequences encoding human,rat and canine CH3 domain of an IgE molecule disclosed in GenbankAccession Numbers S53497, X00923, and L36872; respectively, areincorporated herein by reference.

[0048] The present invention also encompasses antigenic fusion proteinscomprising an antigenic peptide of the invention encoded by apolynucleotide sequence from two different species and a heterologouscarrier protein encoded by a polynucleotide sequence of a differentspecies from the antigenic peptide. The polynucleotide sequence of aheterologous carrier protein can be obtained from scientific literature,Genbank, or using cloning techniques known to those of skill in the art.

[0049] The polynucleotide sequence encoding an antigenic peptide or anantigenic fusion protein of the invention can be inserted into anappropriate expression vector, i.e., a vector, which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. The necessary transcriptional and translationalsignals can also be supplied by the native IgE genes or its flankingregions. A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

[0050] Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining polynucleotides encoding antigenic peptides or antigenicfusion proteins, and appropriate transcriptional and translationalcontrol signals. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of the nucleic acid sequence encoding an antigenic peptide oran antigenic fusion protein of the invention may be regulated by asecond nucleic acid sequence so that the antigenic peptide or theantigenic fusion protein is expressed in a host transformed with therecombinant DNA molecule. For example, expression of an antigenicpeptide or an antigenic fusion protein of the invention may becontrolled by any promoter or enhancer element known in the art.Promoters which may be used to control the expression of an antigenicpeptide or an antigenic fusion protein of the invention include, but arenot limited to, the Cytomeglovirus (CMV) immediate early promoterregion, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290: 304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296: 39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731),or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242: 74-94; plant expression vectorscomprising the nopaline synthetase promoter region (Herrera-Estrella etal., Nature 303: 209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310: 115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald,1987, Hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122);immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature318: 533-538; and Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45: 485-495); albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1: 268-276); alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5: 1639-1648; and Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in theliver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315: 338-340; and Kollias et al., 1986, Cell 46: 89-94);myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active inskeletal muscle (Sani, 1985, Nature 314: 283-286); swine alpha-skeletalactin control region which is active in muscle (Reecy, M. et al., 1998,Animal Biotechnology 9: 101-120) ;and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234: 1372-1378).

[0051] In a specific embodiment, a vector is used that comprises apromoter operably linked to an antigenic peptide-encoding nucleic acid,one or more origins of replication, and, optionally, one or moreselectable markers (e.g., an antibiotic resistance gene). In anotherspecific embodiment, a vector is used that comprises a promoter operablylinked to an antigenic fusion protein-encoding nucleic acid, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

[0052] Expression vectors containing gene inserts can be identified bythree general approaches: (a) nucleic acid hybridization; (b) presenceor absence of “marker” gene functions; and (c) expression of insertedsequences. In the first approach, the presence of antigenicpeptide-encoding polynucleotides or antigenic fusion protein-encodingpolynucleotides inserted in an expression vector(s) can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to the inserted polynucleotide sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of the gene(s) in the vector(s). For example, ifa nucleic acid molecule encoding an antigenic peptide or an antigenicfusion protein is inserted within the marker gene sequence of thevector, recombinants containing the nucleic acid molecule encoding theantigenic peptide or the antigenic fusion protein insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe gene product expressed by the recombinant. Such assays can be based,for example, on the physical or functional properties of an antigenicpeptide or an antigenic fusion protein in in vitro assay systems, e.g.,binding of an antigenic peptide or an antigenic fusion protein with ananti-IgE antibody.

[0053] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

[0054] The term “host cell” as used herein refers not only to theparticular subject cell into which a recombinant DNA molecule isintroduced but also to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

[0055] A host cell strain may be chosen which modulates the expressionof the inserted sequences, or modifies and processes the gene product inthe specific fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers; thus, expression of thegenetically engineered may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce an unglycosylated core protein product.Expression in yeast will produce a glycosylated product. Expression inmammalian cells can be used to ensure “native” glycosylation of anantigenic peptide or antigenic fusion protein of the invention.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

[0056] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress an antigenic peptide or an antigenic fusion protein of theinvention may be engineered. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines which express an antigenic peptide or anantigenic protein of the invention. Such engineered cell lines may beparticularly useful in the screening and evaluation of anti-IgEantibodies or other agents (e.g., organic molecules, inorganicmolecules, organic/inorganic complexes, polypeptides, peptides, peptidemimics, polysaccharides, saccharides, glycoproteins, nucleic acids, DNAand RNA strands and oligonucleotides, etc.) that affect binding of anIgE molecule to its receptor.

[0057] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistanceto hygromycin (Santerre et al., 1984, Gene 30: 147) genes.

[0058] In a specific embodiment, one or more nucleic acid moleculescomprising a polynucleotide sequence encoding an antigenic peptide ofthe invention, are administered to treat or prevent IgE-mediatedallergic disorders, by way of gene therapy. In another specificembodiment, one or more nucleic acid molecules comprising apolynucleotide sequence encoding an antigenic fusion protein, areadministered to treat or prevent IgE-mediated allergic disorders, by wayof gene therapy. In yet another specific embodiment, one or more nucleicacid molecules comprising a polynucleotide sequence encoding anantigenic peptide of the invention, and one or more nucleic acidmolecules comprising a polynucleotide sequence encoding an antigenicfusion protein of the invention are administered to treat or preventIgE-mediated allergic disorders, by way of gene therapy. Gene therapyrefers to therapy performed by the administration to a subject of anexpressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded antigenic peptides orantigenic fusion proteins that mediate a therapeutic effect by elicitingan immune response such as the production of anti-IgE antibodies.

[0059] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0060] For general reviews of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596;Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11(5):155-215).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler,1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,NY.

[0061] In a preferred aspect, a pharmaceutical composition comprisesnucleic acid sequences encoding an antigenic peptide of the invention,said nucleic acid sequences being part of expression vectors thatexpress the antigenic peptide in a suitable host. In particular, suchnucleic acid sequences have promoters operably linked to the antigenicpeptide coding regions, said promoters being inducible or constitutive,and, optionally, tissue-specific. In another preferred aspect, apharmaceutical composition comprises nucleic acid sequences encoding anantigenic fusion protein of the invention, said nucleic acid sequencesbeing part of expression vectors that express the antigenic fusionprotein in a suitable host. In particular, such nucleic acid sequenceshave promoters operably linked to the antigenic fusion protein codingregions, said promoters being inducible or constitutive, and,optionally, tissue-specific. In another particular embodiment, nucleicacid molecules are used in which the coding sequences of an antigenicpeptide of the invention and any other desired sequences are flanked byregions that promote homologous recombination at a desired site in thegenome, thus providing for intrachromosomal expression of the nucleicacids encoding the antigenic peptide (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature342:435-438). In another particular embodiment, nucleic acid moleculesare used in which the coding sequences of an antigenic fusion protein ofthe invention and any other desired sequences are flanked by regionsthat promote homologous recombination at a desired site in the genome,thus providing for intrachromosomal expression of the nucleic acidsencoding the antigenic protein.

[0062] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0063] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.); and WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).

[0064] In specific embodiments, viral vectors that contain nucleic acidsequences encoding antigenic peptides or antigenic fusion proteins areused. For example, a retroviral vector containing nucleic acid sequencesencoding an antigenic peptide or an antigenic fusion protein can be used(see, e.g., Miller et al., 1993, Meth. Enzymol. 217: 581-599). Theseretroviral vectors have been to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The nucleic acid sequences encoding antigenic peptides orantigenic fusion proteins to be used in gene therapy are cloned into oneor more vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6: 291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.3:110-114.

[0065] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3: 499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5: 3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91: 225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2: 775-783. In apreferred embodiment, adenovirus vectors are used. Adeno-associatedvirus (AAV) has also been proposed for use in gene therapy (see, e.g,Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300; and U.S.Pat. No. 5,436,146).

[0066] Another approach to gene therapy involves transferring a nucleicacid molecule to cells in tissue culture by such methods aselectroporation, lipofection, calcium phosphate mediated transfection,or viral infection. Usually, the method of transfer includes thetransfer of a selectable marker to the cells. The cells are then placedunder selection to isolate those cells that have taken up and areexpressing the transferred gene. Those cells are then delivered to apatient.

[0067] In this embodiment, the nucleic acid molecule is introduced intoa cell prior to administration in vivo of the resulting recombinantcell. Such introduction can be carried out by any method known in theart, including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign nucleic acid molecules into cells (see, e.g., Loeffler and Behr,1993, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol.217: 618-644; Cline, 1985, Pharmac. Ther. 29: 69-92) and may be used inaccordance with the present invention, provided that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted. The technique should provide for the stable transfer of thenucleic acid to the cell, so that the nucleic acid is expressible by thecell and preferably heritable and expressible by its cell progeny.

[0068] The resulting recombinant cells can be delivered to a subject byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, subject's state, etc., and can be determined by oneskilled in the art.

[0069] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0070] In a preferred embodiment, the cell used for gene therapy isautologous to the subject.

[0071] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding the antigenic peptides orantigenic fusion proteins of the invention are introduced into the cellssuch that they are expressible by the cells or their progeny, and therecombinant cells are then administered in vivo for therapeutic effect.In a specific embodiment, stem or progenitor cells are used. Any stemand/or progenitor cells which can be isolated and maintained in vitrocan potentially be used in accordance with this embodiment of thepresent invention (see e.g., PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71: 973-985; Rheinwald, 1980,Meth. Cell Bio. 21A: 229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61: 771).

[0072] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0073] The invention also relates to methods for producing an antigenicpeptide of the invention or an antigenic fusion protein of the inventioncomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing anantigenic peptide or an antigenic fusion protein of the invention inwhich a host cell (i.e., a prokaryotic or eukaryotic cell) containing asuitable expression vector that includes a polynucleotide encoding anantigenic peptide or an antigenic fusion protein is cultured underconditions that allow expression of the encoded antigenic peptide or theencoded antigenic fusion protein. The antigenic peptide or the antigenicfusion protein can be recovered from the culture, conveniently from theculture medium, and further purified. The purified antigenic peptides orantigenic fusion proteins can be used in in vitro immunoassays which arewell known in the art to identify anti-IgE antibodies which bind to theantigenic peptides or the antigenic fusion proteins.

[0074] The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods arewell known in the art, as described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), incorporatedherein by reference. As used herein, an insect cell capable ofexpressing a polynucleotide of the present invention is “transformed.”

[0075] Alternatively, an antigenic peptide of the invention or anantigenic fusion protein of the invention may also be expressed in aform which will facilitate purification. For example, an antigenicpeptide may be expressed as fusion protein comprising a heterologousprotein such as maltose binding protein (MBP) glutathione-S-transferase(GST) or thioredoxin (TRX) which facilitate purification. Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and In Vitrogen, respectively. The protein can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.).

[0076] The antigenic peptides of the invention or the antigenic fusionproteins of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the antigenic peptide or theantigenic fusion protein.

[0077] Any method known to those of skill in the art can be used toproduce an antigenic peptide or an antigenic fusion protein of theinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. The isolated antigenic peptides and antigenic fusionproteins of the invention are useful, for example, in generatingantibodies against the native polypeptide.

[0078] One skilled in the art can readily follow known methods forisolating peptides and proteins in order to obtain one of the isolatedantigenic peptides or antigenic fusion proteins of the presentinvention. These include, but are not limited to, immunochromatography,high performance liquid chromatography (HPLC), reverse-phase highperformance liquid chromatography (RP-HPLC), size-exclusionchromatography, ion-exchange chromatography, and immuno-affinitychromatography. See, e.g., Scopes, Protein Purification: Principles andPractice, Springer-Verlag (1994); Sambrook et al., in Molecular Cloning:A Laboratory Manual; Ausubel et al., Current Protocols in MolecularBiology.

[0079] An antigenic peptide or an antigenic fusion protein of theinvention is “isolated” or “purified” when it is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, protein that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of a contaminating protein. When an antigenicpeptide or an antigenic fusion protein of the invention is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When an antigenic peptide or anantigenic fusion protein of the invention is produced by chemicalsynthesis, it is preferably substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the antigenic peptideor the antigenic fusion protein. Accordingly such preparations of theprotein have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or compounds other than the antigenic peptide or theantigenic fusion protein.

[0080] The compositions of the invention are preferably tested in vitro,and then in vivo for the desired therapeutic or prophylactic activity,prior to use in humans. For example, in vitro assays which can be usedto determine whether administration of a specific composition isindicated, include in vitro cell culture assays in which a patienttissue sample is grown in culture, and exposed to or otherwiseadministered a composition, and the effect of such composition upon thetissue sample is observed.

[0081] The expression of an antigenic peptide or an antigenic fusionprotein can be assayed by the immunoassays, gel electrophoresis followedby visualization, or any other method known to those skilled in the art.

[0082] In various specific embodiments, in vitro assays can be carriedout with representative cells of cell types involved in a patient'sdisorder, to determine if a composition has a desired effect upon suchcell types. In accordance with the present invention, the functionalactivity of an antigenic peptide or an antigenic fusion protein can bemeasured by its ability to induce anti-IgE antibodies that inhibit IgEfrom binding to its receptor on mast cells or basophils in vitro withoutinducing the release of vasoactive substances such as histamine.

[0083] Compositions for use in therapy can be tested in suitable animalmodel systems prior to testing in humans, including but not limited topigs, chicken, cows or monkeys.

[0084] The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a composition ofthe invention to elicit the production of anti-IgE antibodies which donot cause anaphylaxis. In a preferred aspect, a composition of theinvention is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

[0085] Formulations and methods of administration that can be employedwhen the composition comprises a nucleic acid are described above;additional appropriate formulations and routes of administration can beselected from among those described herein below.

[0086] Various delivery systems are known and can be used to administera composition of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe composition, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262: 4429-4432), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intratumoral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent.

[0087] In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, topical application, injection, or by meansof an implant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of an allergic reaction.

[0088] In another embodiment, a composition of the invention can bedelivered in a vesicle, in particular a liposome (see, e.g., Langer,1990, Science 249: 1527-1533; Treat et al., in Liposomes in the Therapyof Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),Liss, New York, pp. 353-365 (1989); and Lopez-Berestein, ibid., pp.317-327; see generally ibid.)

[0089] In yet another embodiment, a composition of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see, e.g., Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; and Saudek et al.,1989, N. Engl. J. Med. 321: 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985,Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; and Howardet al.,1989, J. Neurosurg. 71: 105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

[0090] Other controlled release systems are discussed in the review byLanger (1990, Science 249: 1527-1533).

[0091] In a specific embodiment where the composition of the inventionis a nucleic acid encoding an antigenic peptide or an antigenic fusionprotein of the invention, the nucleic acid can be administered in vivoto promote expression of its encoded antigenic peptide or antigenicfusion protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.USA 88: 1864-1868), etc. Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination.

[0092] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of anantigenic peptide or an antigenic fusion protein of the invention, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantigenic peptide or the antigenic fusion protein, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0093] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0094] The antigenic peptides or antigenic fusion proteins of theinvention can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

[0095] The following examples further illustrate the invention.

EXAMPLES 1. Selection of Antigenic Peptides and Cloning of CorrespondingPolyneucleotide Sequences

[0096] A major obstacle facing the development of an anti-IgE vaccine isthe lack of information regarding the precise amino acids representingnon-anaphylactogenic IgE determinants that could be safely used toimmunize allergic subjects and induce non-anaphylactogenic polyclonalantibodies (i.e. polyclonal anti-IgE antibodies that do not bind toreceptor-bound IgE). By definition, those determinants ideallycorrespond to only those IgE amino acid sequences that physicallycontact the IgE receptor. Clearly, there is no information in the priorart on the precise identity of those sequences. Indeed, the prior art isinconsistent on even the region or domain of IgE within which thosesequences may reside. Furthermore, the identity of non-anaphylactogenicdeterminants could be correctly elucidated from only solving the crystalstructure of IgE-IgE receptor complex which, unfortunately, has not yetbeen achieved. Consequently, the present invention overcome thisobstacle and provide IgE determinants capable of inducing withinallergic hosts therapeutically desirable polyclonal antibodies thatreact with native serum IgE, prevent IgE from binding to its receptor onmast cells and basophils and do not react with receptor-bound IgE. Inorder to identify non-anaphylactogenic IgE epitopes suitable forinclusion into an anti-IgE vaccine, we follow an approach that does notmake any a priori assumptions about the location or require knowledge ofthe exact amino acids on IgE suitable for an effective and safe vaccine.The IgE antibody has been to shown to exist in many species throughoutthe animal kingdom including for example humans, dogs, cats, sheep,cows, pigs, horses, rats, mice and chimpanzee. Indeed the IgE gene andits encoded protein have been identified in all these species.Comparison of the primary amino acid sequence among these IgE moleculesshows that they share common amino acid sequences in many locationsthroughout the IgE molecule. These common (conserved) sequences areflanked by amino acid sequences that vary among the various IgEmolecules. We reasoned that a comparison of the primary sequence of IgEmolecules from different species e.g. rat IgE and dog IgE would provideclues to identification of non-anaphylactogenic IgE determinants. It isknown that dog, cat, and rat IgE bind to the human IgE receptor. SinceIgE from dog and rat bind to the IgE receptor of another unrelatedspecies such as human receptor, we hypothesize that the conserved aminoacids between rat and dog must contain the information specifying thereceptor-binding conformational determinants. Since these conservedsequences are flanked within their respective IgE molecule withsequences that vary between dog and rat IgE, we further hypothesize thatthe variable IgE sequences could be exchanged between IgE from dog andrat without affecting the overall receptor-binding conformation ofeither IgE molecules. Using this reasoning, a safe and effective vaccinefor dogs could be developed by using peptides of the present inventionSEQ ID NOS: 1 to 6. The nucleotide sequences encoding antigenic peptidesof the present invention were prepared using the following procedures:

[0097] Cloning of Dog CH3 Domain (SEQ ID NO: 15).

[0098] The polyneucleotide sequence encoding dog CH3 domain wasassembled by a polymerase chain reaction (PCR)-based gene synthesisprocedure. A set of oligonucleotide primers (listed in Table 1) wassynthesized at Life Technologies Inc. TABLE 1 Primers for cloning of DogCH3 domain DNA (SEQ ID NO:15) Primer Primer sequence nameAAGCGTGCCCCCCCGGAAGTCTATGCGTTTGCGAC P173-S712TCGGGGGTCGGACTCTGAACACTTCTTGGTGCTGTC P173-A402GACAGCACCAAGAAGTGTTCAGAGTCCGACCCCCGAGGCGT P173-S1 GACGAGCTACCTGAGCCCACCCAGCCCCCTTGACCTGTATGTC P173-S2CACAAGGCGCCCAAGATCACCTGCCTGGTAGTGGACCTGG P173-53CCACCATGGAAGGCATGAACCTGACCTGGTACCGGGAGAG P173-S4CAAAGAACCCGTGAACCCGGGCCCTTTGAACAAGAAGGAT P173-S5CACTTCAATGGGACGATCACAGTCACGTCTACCCTGCCAG P173-S6TGAACACCAATGACTGGATCGAGGGCGAGACCTACTATTG P173-S7CAGGGTGACCCACCCGCACCTGCCCAAGGACATCGTGCGC P173-S8TCCATTGCCAAGGCCCCTGGCAAGCGTGCCCCCCCGGAAG P173-S9CGGCGTCGCAAACGCATAGACTTCCGGGGGGGCACGCTTG P173-A1CCAGGGGCCTTGGCAATGGAGCGCACGATGTCCTTGGGCA P173-A2GGTGCGGGTGGGTCACCCTGCAATAGTAGGTCTCGCCCTC P173-A3GATCCAGTCATTGGTGTTCACTGGCAGGGTAGACGTGACT P173-A4GTGATCGTCCCATTGAAGTGATCCTTCTTGTTGAAAGGGC P173-A5CCGGGTTCACGGGTTCTTTGCTCTCCCGGTACCAGGTCAG P173-A6GTTCATGCCTTCCATGGTGGCCAGGTCCACTACCAGGCAG P173-A7GTGATCTTGGGCGCCTTGTGGACATACAGGTCAAGGGGGC P173-A8TGGGTGGGCTCAGGTAGCTCGTCACGCCTCGGGGGTCGGA P173-A9

[0099] These primers were used to assemble the dog CH3 domain in atwo-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 18 primers (P173-S1 to -S9 and P173-A1 to -A9) followed by 2)dilution of the product from step 1 (0.625 ul into a 50 ul reaction)into a new reaction and carrying out 15 cycles of PCR using the twoterminal primers (P173-S1 and P173-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize.

[0100] Cloning of Nucleotide Sequence Encoding Partial Human CH3/PartialDog CH3 Domain Fusion Protein (SEQ ID NO: 16).

[0101] This DNA sequence encodes a protein which consists of the first63 amino acids of human CH3 domain fused to the last 53 amino acid ofthe dog CH3 domain. The polyneucleotide sequence encoding this constructwas assembled as follows: A set of oligonucleotide primers (listed intable 2) was synthesized at Life Technologies Inc. TABLE 2 Primers forcloning of human/dog CH3 domain fusion DNA (SEQ ID NO:16) Primer Primersequence name CCTCTCGGGTTGGAATCTGCACACTTCTTGGTGCTGTC P174-A404AAGGGTGCCCCCCCGGAAGTCTATGCGTTTGCGAC P174-5721GACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P174-S1 GAGCGCCTACCTAAGCCGGCCCAGCCCGTTCGACCTGTTCATC P174-S2CGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTGG P174-53CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P174-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAAGGAGGAG P174-55AAGAAGGATCACTTCAATGGGACGATCACAGTCACGTCTA P174-56CCCTGCCAGTGAACACCAATGACTGGATCGAGGGCGAGAC P174-57CTACTATTGCAGGGTGACCCACCCGCACCTGCCCAAGGAC P174-58ATCGTGCGCTCCATTGCCAAGGCCCCTGGCAAGCGTGCCC P174-59AAACGCATAGACTTCCGGGGGGGCACGCTTGCCAGGGGCC P174-A1TTGGCAATGGAGCGCACGATGTCC1TGGGCAGGTGCGGGT P174-A2GGGTCACCCTGCAATAGTAGGTCTCGCCCTCGATCCAGTC P174-A3ATTGGTGTTCACTGGCAGGGTAGACGTGACTGTGATCGTG P174-A4CCATTGAAGTGATCCTTCTTCTCCTCCTTTCTGGTGGAGT P174-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P174-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P174-A7GTGATCGTGGGCGACTTGCGGATGAACAGGTCGAACGGGC P174-A8TGGGCCGGCTTAGGTAGGCGCTCACCCCTCTCGGGTTGGA P174-A9

[0102] These primers were used to assemble the human CH3/dog CH3 domainfusion in a two-step PCR reaction as follows: 1) 25 cycles using anequimolar mixture of 18 primers (P174-S1 to -S9 and P174-A1 to -A9)followed by 2) dilution of the product from step 1 (0.625 μl into a 50μl reaction) into a new reaction and carrying out 15 cycles of PCR usingthe two terminal primers (P174-S1 and P174-A1). All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size (384 nucleotides).

[0103] Cloning of Chimeric Human/Dog CH3 domain (SEQ ID NO: 17).

[0104] This DNA sequence encodes a protein, which consists ofalternating human/dog CH3 domain sequences. The polyneucleotide sequenceencoding human CH3/conserved dog CH3 domain was assembled as follows: Aset of oligonucleotide primers (listed in Table 3) was synthesized atLife Technologies Inc. TABLE 3 Primers for cloning of human/dog CH3domain chimeric DNA (SEQ ID NO:17) Primer Primer sequence nameGACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P175-S1 GACCAGCTACCTAAGCCCGCCCAGCCCGCTGGACCTGTACATC P175-52CGCAAGTCGCCCAAGATCACCTGTCTGGTGGTGGACCTGG P175-53CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P175-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAAGGAGGAG P175-S5AAGCAACGGAATGGGACGATCACAGTCACGTCTACCCTGC P175-S6CAGTGGGCACCAGAGACTGGATCGAGGGCGAGACCTACTA P175-S7TTGCAGGGTGACCCACCCGCACCTGCCCAAGGACATCGTG P175-S8CGCTCCATTGCCAAGGCCCCTGGCAAGCGTGCCCCCCCGG P175-S9CGTCGCAAACGCATAGACTTCCGGGGGGGCACGCTTGCCA P175-A1GGGGGCTTGGCAATGGAGCGCACGATGTCCTTGGGCAGGT P175-A2GCGGGTGGGTCACCCTGCAATAGTAGGTCTCGCCCTGGAT P175-A3CCAGTCTCTGGTGCCCACTGGCAGGGTAGACGTGACTGTG P175-A4ATCGTCCCATTCCGTTGCTTCTCCTCCTTTCTGGTGGAGT P175-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P175-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P175-A7GTGATCTTGGGCGACTTGCGGATGTACAGGTCCAGCGGGC P175-A8TGGGCGGGCTTAGGTAGCTGGTCACCCCTCTCGGGTTGGA P175-A9CCTCTCGGGTTGGAATCTGCACACTTCTTGGTGCTGTCCT P175-A404AAGCGTGCCCCCCCGGAAGTCTATGCGTTTG P175-5715

[0105] These primers were used to assemble the human CH3/dog CH3chimeric domain in a two-step PCR reaction as follows: 1) 25 cyclesusing an equimolar mixture of 18 primers (P175-S1 to -S9 and P175-A1 to-A9) followed by 2) dilution of the product from step 1 (0.625 ul into a50 ul reaction) into a new reaction and carrying out 15 cycles of PCRusing the two terminal primers (P175-S1 and P175-A1). All reactions usedBMB Expand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size (384 nucleotides).

[0106] Cloning of HumanCH3 Domain (SEQ ID NO: 18).

[0107] The polyneucleotide sequence encoding human CH3 domain wasassembled as follows: A set of oligonucleotide primers (listed in Table4) was synthesized at Life Technologies Inc. TABLE 4 Primers for HumanCH3 domain DNA (SEQ ID NO:18) Primer Primer sequence nameGACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P176-S1 GAGCGCCTACCTAAGCCGGCCCAGCCCGTTCGACCTGTTCATC P176-S2CGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTGG P176-S3CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P176-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAAGGAGGAG P176-S5AAGCAGCGCMTGGCACGTTAACCGTCACGTCCACCCTGC P176-S6CGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCA P176-S7GTGCAGGGTGACCCACCCCCACCTGCCCAGGGCCCTCATG P176-S8CGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCGG P176-S9CGTCGCAAACGCATAGACTTCCGGGGCAGCACGCGGGCCG P176-A1CTGGTCTTGGTCGTGGACCGCATGAGGGCCCTGGGCAGGT P176-A2GGGGGTGGGTCACCCTGCACTGGTAGGTCTCCCCCTCGAT P176-A3CCAGTCTCGGGTGCCCACCGGCAGGGTGGACGTGACGGTT P176-A4AACGTGCCATTGCGCTGCTVCTCCTCCTTTCTGGTGGAGT P176-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P176-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P176-A7GTGATCGTGGGCGACTTGCGGATGAACAGGTCGAACGGGC P176-A8TGGGCCGGCTTAGGTAGGCGCTCACCCCTCTCGGGTTGGA P176-A9CCTCTGGGGTTGGAATCTGCACACTTCTTGGTGCT P176-A404GCGGCCCGCGTGCTGCCCCGGAAGTCTATGCGTTTGCGAC P176-S710

[0108] These primers were used to assemble the human CH3 domain in atwo-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 18 primers (P176-S1 to -S9 and P176-A1 to -A9) followed by 2)dilution of the product from step 1 (0.625 ul into a 50 ul reaction)into a new reaction and carrying out 15 cycles of PCR using the twoterminal primers (P176-S1 and P176-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (384 nucleotides).

[0109] Cloning of Rat CH3 Domain (SEQ ID NO: 19).

[0110] The polyneucleotide sequence encoding rat CH3 domain wasassembled as follows: A set of oligonucleotide primers (listed in Table5) was synthesized at Life Technologies Inc. TABLE 5 Primers for Rat CH3DNA (SEQ ID NO:19) Primer Primer sequence nameGACAGCACCAAGAAGTGCTCAGATGATGAGCCCCGGGGTGT P177-S1 GATTACCTACCTGATCCCACCCAGTCCCCTCGACCTGTATGAA P177-S2AATGGGACTCCCAAACTTACCTGTCTGGTTTTGGACCTGG P177-S3AAAGTGAGGAGAATATCACCGTGACGTGGGTCCGAGAGCG P177-S4TAAGAAGTCTATAGGTTCGGCATCCCAGAGGAGTACCAAG P177-S5CACCATAATGCCACAACCAGTATCACCTCCATCTTGCCAG, P177-S6TGGATGCCAAGGACTGGATCGAAGGTGAAGGCTACCAGTG P177-S7CAGAGTGGACCACCCTCACTTTCCCAAGCCCATTGTGCGT P177-S8TCCATCACCAAGGCCCCAGGCAAGCGCTCAGCCCCAGAAG P177-S9CGGCGTCGCAAACGCATAGACTTCTGGGGCTGAGCGCTTG P177-A1CCTGGGGCCTTGGTGATGGAACGCACAATGGGCTTGGGAA P177-A2AGTGAGGGTGGTCCACTCTGCACTGGTAGCCTTCACCTTC P177-A3GATCCAGTCCTTGGCATCCACTGGCAAGATGGAGGTGATA P177-A4CTGGTTGTGGCATTATGGTGCTTGGTACTCCTCTGGGATG P177-A5CCGAACCTATAGACTTCTTACGCTCTCGGACCCACGTCAC P177-A6GGTGATATTCTCCTCACTTTCCAGGTCCAAAACCAGACAG P177-A7GTAAGTTTGGGAGTCCCATTTTCATACAGGTCGAGGGGAC P177-A8TGGGTGGGATCAGGTAGGTAATCACACCCCGGGGCTCATC P177-A9CGGGGCTCATCATCTGAGCACTTCTTGGTGCTGTCCT P177-A401CAAGCGCTCAGCCCCAGAAGTCTATGCGTTTGCGAC P177-5711

[0111] These primers were used to assemble the rat CH3 domain in atwo-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 18 primers (P177-S1 to -S9 and P177-A1 to -A9) followed by 2)dilution of the product from step 1 (0.625 μl into a 50 μl reaction)into a new reaction and carrying out 15 cycles of PCR using the twoterminal primers (P177-S1 and P177-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (384nucleotides).

[0112] Cloning of Human CH3 for Expression in Baculovirus (SEQ ID NO:20).

[0113] The polyneucleotide sequence encoding the IgE CH3 domain and thesignal sequence from honey-bee mellitin was assembled as follows: A setof oligonucleotide primers (listed in Table 6) was synthesized at LifeTechnologies Inc. TABLE 6 Primers for baculovirus expressed Human IgECH3 domain (SEQ ID NO:20) Primer Primer sequence nameGCGGATCCATGAAATTCTTAGTCAACGTTGCCCTTGTTTTAT P158-S1GGTCGTATACATTTCTTACATCTATGCGGACAGCAACCCG P158-S2AGAGGGGTGAGCGCCTACCTAAGCCGGCCCAGCCCGTTCG P158-S3ACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGT P158-S4GGTGGACCTGGCACCCAGCAAGGGGACCGTGAACCTGACC P158-S5TGGTCCCGGGCCAGTGGGAAGCCTGTGAACCACTCCACCA P158-S6GAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCAC P158-S7GTCCACCCTGCCGGTGGGCACCCGAGACTGGATCGAGGGG P158-S8GAGACCTACCAGTGCAGGGTGACCCACCCCCACCTGCCCA P158-S9GGGCCCTCATGCGGTCCACGACCAAGACCTCCTGATGAATTC P158-S10 CGG P158-A1CCGGAATTCATCAGGAGGTCTTTGGT P158-A2CGTGGACCGCATGAGGGCCCTGGGCAGGTGGGGGTGGGTC P158-A3ACCCTGCACTGGTAGGTCTCCCCCTCGATCCAGTCTCGGG P158-A4TGCCCACCGGCAGGGTGGACGTGACGGTTAACGTGCCATT P158-ASGCGCTGCTTCTCCTCCTTTCTGGTGGAGTGGTTCACAGGC P158-A6TTCCCACTGGCCCGGGACCAGGTCAGGTTCACGGTCCCCT P158-A7TGCTGGGTGCCAGGTCCACCACCAGACAGGTGATCGTGGG P158-A8CGACTTGCGGATGAACAGGTCGAACGGGCTGGGCCGGCTT P158-A9AGGTAGGCGCTCACCCCTCTCGGGTTGCTGTCCGCATAGA P158-S10TGTAAGAAATGTATACGACCATAAAAACAAGGGCAACGTT

[0114] These primers were used to assemble the human CH3 domain in atwo-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 20 primers (P158-S1 to -S10 and P158-A1 to -A10) followed by2) dilution of the product from step 1 (1.25 ul into a 50 ul reaction)into a new reaction and carrying out 10 cycles of PCR using primers(P158-S1 and P158 A1). All reactions used BMB Expand HF polymerasemixture and conditions specified by the manufacturer. This PCR reactionresulted in amplification of a gene sequence of the correct size (400nucleotides). The PCR amplified 409 bp fragment was digested with EcoRIand BamHI enzymes and ligated to pFastBac1 plasmids digested with EcoRIand BamHI enzymes. The ligation mixture was transformed into DH5 E. coliand colonies containing the plasmid plus 409 bp insert were isolated.Plasmid DNA was prepared from DH5 cells using Quiagen columns accordingto the manufacturer's recommendation. The “donor plasmid”(pFastBac1-CH3) DNA was transformed into DH10Bac competent cells fortransposition into the bacmid according to the Bac-To-Bac BaculovirusExpression System protocol (Life Technologies). White colonies thatcontained the recombinant bacmid were isolated and grown up forisolation of bacmid DNA. To isolate bacmid DNA, Concert High PurityPlasmid Isolation System (Life Technologies) was used according to themethods provided by the manufacturer. PCR analysis of recombinant bacmidwas used to confirm that the CH3 gene had transposed into the bacmid.PUC/M13 amplification primers (Life Technologies) were used in reactionconditions specified by the Bac-To-Bac Expression Systems manual. Thereaction yielded a single specific product 2709 base pairs in sizeindicating that the CH3 domain gene was inserted into the bacmid (bacmidtransposed with pFastBac1 2300 bp+CH3 domain 409 bp=2709 bp).

[0115] 2. Selection of Protein Carrier and Cloning of CorrespondingPolyneucleotide Sequences.

[0116] The antigenic peptides of the present invention were incorporatedwithin a carrier protein whose function is to increase theimmunogenicity of the peptides and at the same time preserve theconformational attributes of these peptides that are necessary to inducethe appropriate anti-IgE antibodies. For this purpose, a carrier systemwas developed based on the utilization of a modified CH2 and CH4 domainsof human IgE. The modification of human CH2 and CH4 domain wereintroduced so as to avoid immunological cross-reactivity between humanCH2-CH4 protein sequence and dog CH2-CH4 protein sequence. The aminoacid sequence of the carrier protein SEQ ID NO: :7-9 was cloned usingthe following procedures:

[0117] Cloning of Human CH2 Domain (SEQ ID NO: 21):

[0118] The polynucleotide sequence encoding signal sequence::human CH2domain was assembled by a two step polymerase chain reaction (PCR)-basedgene synthesis procedures follows: A set of oligonucleotide primers(listed in Table 7) was synthesized at Life Technologies Inc. TABLE 7Primers for Human CH2 domain DNA (SEQ ID NO:21) Primer Primer sequencename GACTGCTAGCCATGAGTGTGCCCA P171-S1bGACTGCTAGCCATGAGTGTGCCCACTCAGGTCCTGGGGTT P171-S1GCTGCTGCTGTGGCTTACAGATGCCAGATGTGACATCGTC P171-S2GCCTCCAGGGACTTCACCCCGCCCTCCGTGAAGATCTTAC P171-S3AGTCGTCCTGCGACGGCGGCGGGCACTTCCCCCCGACCAT P171-S4CCAGCTCTACTGCCTCGTCTCTGGGTACACCCCAGGGACT P171-S5ATCCAGATCACCTGGCTGGAGGACGGGCAGGTCATGGACG P171-S6TGGACTTGTCCACCGCCTCTACCACGCAGGAGGGTGAGCT P171-S7GGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC P171-S8TGGCTGTCAGACCGCACCTTCACCTGCCAGGTCACCTATC P171-S9AAGGTCACACCTTTGAGGACAGCACCAAGAAGTGTCTCGA P171-S10GACTCTCGAGACACTTCTTGGTGCT P171-A1GTCCTCAAAGGTGTGACCTTGATAGGTGACCTGGCAGGTG P171-A2AAGGTGCGGTCTGACAGCCAGTGCTTCTGGCTGAGGGTGA P171-A3GCTCGCTTTGTGTGGAGGCCAGCTCACCCTCCTGCGTGGT P171-A4AGAGGCGGTGGACAAGTCCACGTCCATGACCTGCCCGTCC P171-A5TCCAGCCAGGTGATCTGGATAGTCCCTGGGGTGTACCCAG P171-A6AGACGAGGCAGTAGAGCTGGATGGTCGGGGGGAAGTGCCC P171-A7GCCGCCGTCGCAGGACGACTGTAAGATCTTCACGGAGGGC P171-A8GGGGTGAAGTCCCTGGAGGCGACGATGTCACATCTGGCAT P171-A9CTGTAAGCCACAGCAGCAGCAACCCCAGGACCTGAGTGGG P171-A10 TGGCTGTCAGACCGCACCTTCAP171-3321 ACTTCTTGGTGCTGTCCTCA P171-A393

[0119] These primers were used to assemble the signal sequence::humanCH2 domain in a two-step PCR reaction as follows: 1) 25 cycles using anequimolar mixture of 20 primers (P171-S1 to -S10 and P171-A1 to -A10)followed by 2) dilution of the product from step 1 (0.625 ul into a 50ul reaction) into a new reaction and carrying out 15 cycles of PCR usingthe two terminal primers (P171-S1b and P171-A1). All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size. The amplified gene was then cloned into(pGEM-T) vector at the T/A cloning site. The nucleotide sequence of theamplified fragment was determined by automated fluorescent DNAsequencing.

[0120] Cloning of Human CH4 Domain (SEQID# 22).

[0121] The polynucleotide sequence encoding human CH4 domain wasassembled by a two step polymerase chain reaction (PCR)-based genesynthesis procedure as follows: A set of oligonucleotide primers (listedin Table 8) was synthesized at Life Technologies Inc. TABLE 8 Primersfor Human CH4 DNA (SEQ ID NO:22) Primer Primer sequence nameGACTCTCGAGGAAGTCTATGCGTT P172-S1bGACTCTCGAGGAAGTCTATGCGTTTGCGACGCCGGAGTGG P172-S1CCGGGGAGCCGGGACAAGCGCACCCTCGCCTGCCTGGTGC P172-S2AGAACTTCATGCCTGAGGACATCTCGGTGCGGTGGGTGCA P172-S3CAACGAGGTGCAGCTCCCGGACGCCCGGCACAGCACGACG P172-S4CAGCCCCGCAAGACCAAGGGCTCCGGCTTCTTCGTCTTCA P172-S5GCCGCCTGGCGGTGACCAGGGCCGAATGGCAGGAGAAAGA P172-S6TGAGTTCATCTGCCGTGCAGTCCATGAGGCAGCGAGCCCC P172-S7TCACAGACCGTCCAGCGAGCGGTGTCTGTAAATCCCGGTA P172-S8GACTGAATTCTCATTTACCGGGATT P172-A1b GACTGAATTCTCATTTACCGGGATTTACAGACACCP172-A1 GCTCGCTGGACGGTCTGTGAGGGGCTCGCTGCCTCATGGA P172-A2CTGCACGGCAGATGAACTCATCTTTCTCCTGCCATTCGGC P172-A3CCTGGTCACCGCCAGGCGGCTGAAGACGAAGAAGCCGGAG P172-A4CCCTTGGTCTTGCGGGGCTGCGTCGTGCTGTGCCGGGCGT P172-A5CCGGGAGCTGCACCTCGTTGTGCAGCCAGCGCACCGAGAT P172-A6GTCCTCAGGCATGAAGTTCTGCACCAGGCAGGCGAGGGTG P172-A7CGCTTGTCCCGGCTCCCCGGCCACTCCGGCGTCGCAAACG P172-A8 GAAGTCTATGCGTTTGCGACGP172-311 GCAGCCAGCGCACCGAGATGTC P172-A119

[0122] These primers were used to assemble the human CH4 domain in atwo-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 16 primers (P172-S1 to -S8 and P172-A1 to -A8) followed by 2)dilution of the product from step 1 (0.625 ul into a 50 ul reaction)into a new reaction and carrying out 15 cycles of PCR using the twoterminal primers (P172-S1b and P172-A1b). All reactions used BMB ExpandHF polymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize. The amplified gene was then cloned into (pGEM-T) vector at the T/Acloning site. The nucleotide sequence of the amplified fragment wasdetermined by automated fluorescent DNA sequencing.

[0123] 3. Cloning of Genes Encoding Fusion Protein Vaccines:

[0124] Polynucleotide sequences encoding fusion proteins for use asvaccines (SEQ ID NO: 24-28) of the present invention were prepared asfollows.

[0125] Cloning of IgE-1 Vaccine Construct (SEQ ID NO: 24):

[0126] The insert in construct IgE-1 consists of the signalsequence-human CH2 domain followed by the dog CH3 domain followed by thehuman CH4 domain. Assembly of the insert for IgE-1 consisted of usingthe signal sequence-human CH2 domain as a template for one PCR reaction,and the human CH4 domain as a template for a second PCR reaction. Inthese two reactions, terminal primers were used to generate regions ofhomology with the dog CH3 domain, so that the two ends of the dog CH3domain DNA fragment would hybridize to the two human domain DNAfragments and the three fragments would serve as “megaprimers” in a PCRreaction. The dog CH3 domain was engineered to contain overlappingsequence on either end to the two human domain fragments (CH2 homologyon the 5′ end and CH4 homology on the 3′ end). Then, the three PCRfragments (human CH2, dog CH3 and human CH4) were mixed in a final PCRreaction utilizing the terminal primers to generate a full-lengthproduct. The procedure is outlined as follows: Fragment 1: (signalsequence-human CH2 domain) 413 bp fragment resulting from amplificationof human CH2 domain with primers P171-S1b and P173-A402; fragment wasamplified in 35 cycles of PCR, followed by gel purification. Allreactions used BMB Expand HF polymerase mixture and conditions specifiedby the manufacturer. Fragment 2: (dog CH3 domain) 384 bp fragmentdescribed above; gel purified Fragment 3: (human CH4 domain) 340 bpfragment resulting from amplification of human CH4 domain with primersP173-S712 and P172-A1b; fragment was amplified in 35 cycles of PCR,followed by gel purification. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. Thethree fragments were added in approximately equimolar amounts to a finalPCR reaction using the two terminal primers P171-S1b and P172-A1b, andcarrying out 35 cycles of PCR. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (1.1 kb). The resulting fragment was digested with EcoR I and NheIand subcloned into the corresponding sites of the plasmid pCI-neo. Thenucleotide sequence of the amplified fragment was determined byautomated fluorescent DNA sequencing.

[0127] Cloning of Construct IgE-2 (SEQ ID NO: 25).

[0128] The insert in construct IgE-2 consists of the signalsequence-human CH2 domain followed by the human CH3/dog domain, followedby the human CH4 domain. Assembly of the insert for IgE-2 consisted ofusing the signal sequence-human CH2 domain as a template for one PCRreaction, and the human CH4 domain as a template for a second PCRreaction. In these two reactions, terminal primers were used to generateregions of homology with the human CH3/dog CH3 domain, so that the twoends of the human CH31dog CH3 domain DNA fragment would hybridize to thetwo human domain DNA fragments and the three fragments would serve as“megaprimers” in a PCR reaction. The human CH3/dog CH3 domain wasengineered to contain overlapping sequence on either end to the twohuman domain fragments (CH2 homology on the 5′ end and CH4 homology onthe 3′ end). Then, the three PCR fragments (human CH2, human CH3/dogCH3, human CH4) were mixed in a final PCR reaction utilizing twoterminal primers to generate a full length product. The procedure isoutlined as follows: Fragment 1: (signal sequence-human CH2 domain) 414bp fragment resulting from amplification of human CH2 domain withprimers P171-S1b and P174-A404; fragment was amplified in 25 cycles ofPCR, followed by gel purification. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer.Fragment 2: (human CH3/dog CH3 domain) 384 bp fragment described above;gel purified Fragment 3: (human CH4 domain) 340 bp fragment resultingfrom amplification of human CH4 domain with primers P174-S721 andP172-A1b; fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. The three gel-purifiedfragments were added in approximately equimolar amounts to a final PCRreaction using the two terminal primers P171-S1b and P172-A1b, andcarrying out 35 cycles of PCR. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (1.1 kb). The resulting fragment was digested with EcoR I and Nhe Iand subcloned into the corresponding sites of the plasmid pCI-neo. Thenucleotide sequence of the amplified fragment was determined byautomated fluorescent DNA sequencing.

[0129] Cloning of IgE-3 Vaccine Construct (SEQ ID NO: 26).

[0130] The insert in construct IgE-3 consists of the signalsequence-human CH2 domain followed by the human CH3/conserved dog CH3sequence, followed by the human CH4 domain. Assembly of the insert forIgE-3 consisted of using the signal sequence-human CH2 domain as atemplate for one PCR reaction, and the human CH4 domain as a templatefor a second PCR reaction. In these two reactions, terminal primers wereused to generate regions of homology with the human CH3/conserved dogCH3 sequence, so that the two ends of the middle (human CH3/conserveddog CH3 domain) DNA fragment would hybridize to the two human domain DNAfragments and the three fragments would serve as “megaprimers” in a PCRreaction. The human CH3/conserved dog CH3 sequence was engineered tocontain overlapping sequence on either end to the two human domainfragments (CH2 homology on the 5′ end and CH4 homology on the 3′ end).Then, the three PCR fragments (human CH2, human CH3/conserved dog CH3,human CH4) were mixed in a final PCR reaction utilizing two terminalprimers to generate a full length product. The procedure is outlined asfollows: Fragment 1: (signal sequence-human CH2 domain) 414 bp fragmentresulting from amplification of human CH2 domain with primers P171-S1band P175-A404; fragment was amplified in 25 cycles of PCR, followed bygel purification. All reactions used BMB Expand HF polymerase mixtureand conditions specified by the manufacturer. Fragment 2: (humanCH3/conserved dog CH3 sequence) 384 bp fragment described above; gelpurified Fragment 3: (human CH4 domain) 340 bp fragment resulting fromamplification of human CH4 domain with primers P175-S715 and P172-A1b;fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. The three gel-purifiedfragments were added in approximately equimolar amounts to a final PCRreaction using the two terminal primers P171-S1b and P172-A1b, andcarrying out 35 cycles of PCR. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (1.1 kb). The resulting fragment was digested with EcoR I and Nhe Iand subcloned into the corresponding sites of the plasmid pCI-neo. Thenucleotide sequence of the amplified fragment was determined byautomated fluorescent DNA sequencing.

[0131] Cloning of IgE-4 Vaccine Construct (SEQ ID NO: 27).

[0132] The insert in construct IgE-4 consists of the signalsequence-human CH2 domain followed by the human CH3 domain followed bythe human CH4 domain. Assembly of the insert for IgE-4 consisted ofusing the signal sequence-human CH2 domain as a template for one PCRreaction, and the human CH4 domain as a template for a second PCRreaction. In these two reactions, terminal primers were used to generateregions of homology with the human CH3 domain, so that the two ends ofthe middle (human CH3 domain) DNA fragment would hybridize to the twoterminal human domain DNA fragments and the three fragments would serveas “megaprimers” in a PCR reaction. The human CH3 domain sequence wasengineered to contain overlapping sequence on either end to the twohuman domain fragments (CH2 homology on the 5′ end and CH4 homology onthe 3′ end). Then, the three PCR fragments (human CH2, human CH3, humanCH4) were mixed in a final PCR reaction utilizing two terminal primersto generate a full length product. The procedure is outlined as follows:Fragment 1: (signal sequence-human CH2 domain) 414 bp fragment resultingfrom amplification of human CH2 domain with primers P171-S1b andP176-A404; fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. Fragment 2: (human CH3 domain)384 bp fragment described above; gel purified Fragment 3: (human CH4domain) 345 bp fragment resulting from amplification of human CH4 domainwith primers P176-S710 and P172-A1b; fragment was amplified in 25 cyclesof PCR, followed by gel purification. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. Thethree gel-purified fragments were added in approximately equimolaramounts to a final PCR reaction using the two terminal primers P171-S1band P172-A1b, and carrying out 35 cycles of PCR. All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size (1.1 kb). The resulting fragment wasdigested with EcoR I and Nhe I and subcloned into the correspondingsites of the plasmid pCI-neo. The nucleotide sequence of the amplifiedfragment was determined by automated fluorescent DNA sequencing.

[0133] Cloning of IgE-5 Vaccine Construct (SEQ ID NO: 28).

[0134] The insert in construct IgE-5 consists of the signalsequence-human CH2 domain followed by the rat CH3 domain followed by thehuman CH4 domain. Assembly of the insert for IgE-5 consisted of usingthe signal sequence-human CH2 domain as a template for one PCR reaction,and the human CH4 domain as a template for a second PCR reaction. Inthese two reactions, terminal primers were used to generate regions ofhomology with the rat CH3 domain, so that the two ends of the middle(rat CH3 domain) DNA fragment would hybridize to the two terminal humandomain DNA fragments and the three fragments would serve as“megaprimers” in a PCR reaction. The rat CH3 domain sequence wasengineered to contain overlapping sequence on either end to the twohuman domain fragments (CH2 homology on the 5′ end and CH4 homology onthe 3′ end). Then, the three PCR fragments (human CH2, rat CH3, humanCH4) were mixed in a final PCR reaction utilizing two terminal primersto generate a full-length product. The procedure is outlined as follows:Fragment 1: (signal sequence-human CH2 domain) 411 bp fragment resultingfrom amplification of human CH2 domain with primers P171-S1b andP177-A401; fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. Fragment 2: (rat CH3 domain)384 bp fragment described above; gel purified Fragment 3: (human CH4domain) 341 bp fragment resulting from amplification of human CH4 domain(IgE-6(3′)) with primers P177-S711 and P172-A1b; fragment was amplifiedin 25 cycles of PCR, followed by gel purification. All reactions usedBMB Expand HF polymerase mixture and conditions specified by themanufacturer. The three gel-purified fragments were added inapproximately equimolar amounts to a final PCR reaction using the twoterminal primers P171-S1b and P172-A1b, and carrying out 35 cycles ofPCR. All reactions used BMB Expand HF polymerase mixture and conditionsspecified by the manufacturer. This PCR reaction resulted inamplification of a gene sequence of the correct size (1.1 kb). Theresulting fragment was digested with EcoR I and Nhe I and subcloned intothe corresponding sites of the plasmid pCI-neo. The nucleotide sequenceof the amplified fragment was determined by automated fluorescent DNAsequencing.

[0135] 4. Transfection, Expression and Reactivity of Vaccines withAnti-canine IgE Antibodies.

[0136] Expression of IgE CH3 Domain in Insect Cells:

[0137] Sf9 cells (Life Technologies) derived from Spodoptera frugiperdawere transfected with the Recombinant Bacmid DNA using Cell FECTINreagent (Life Technologies) following the Bac-To-Bac Expression Systemprotocol. At 72 hours supernates were passaged to fresh subconfluent Sf9cells. At 7 days post infection cytopathic effect (CPE) was evident.Supernates were harvested and stored at 4C protected from light. Sampleswere analyzed by electrophoresis (4-12% Bis-Tris Novex NuPage systemreducing conditions). One of the duplicate gels was stained withcoomassie blue and the other was transferred to PVDF membrane (Novex)using standard western blot transfer method. The membrane was probedwith rabbit #145 RBS-2 polyclonal antiserum followed by AP-rec Protein G(Zymed). A distinct band of approximately 14.5 kDa was evident in boththe coomassie stained gel (FIG. 1) and western blot (FIG. 2) indicatingexpression of the CH3 protein.

[0138] Sequences of the present invention are described in Table 9.TABLE 9 Sequence Listings Protein/ Composition Sequence ID# DNA Dog CH3domain SEQ ID NO: 1 Protein Human/dog CH3 domain fusion SEQ ID NO: 2Protein Human/dog CH3 domain chimera SEQ ID NO: 3 protein Human CH3domain SEQ ID NO: 4 Protein Rat CH3 domain SEQ ID NO: 5 Protein HumanCH3 domain (baculovirus expressed) SEQ ID NO: 6 protein Modified humanCH2 domain SEQ ID NO: 7 Protein Modified human CH4 domain SEQ ID NO: 8Protein human CH2-CH4 carrier protein SEQ ID NO: 9 protein IgE-1 fusionprotein SEQ ID NO: 10 Protein IgE-2 fusion protein SEQ ID NO: 11 ProteinIgE-3 fusion protein SEQ ID NO: 12 protein IgE-4 fusion protein SEQ IDNO: 13 Protein IgE-5 fusion protein SEQ ID NO: 14 protein Dog CH3 domainSEQ ID NO: 15 DNA Human/dog CH3 domain fusion SEQ ID NO: 16 DNAHuman/dog CH3 domain chimera SEQ ID NO: 17 DNA Human CH3 domain SEQ IDNO: 18 DNA Rat CH3 domain SEQ ID NO: 19 DNA Human CH3 domain(baculovirus SEQ ID NO: 20 DNA expressed) Modified human CH2 domain SEQID NO: 21 DNA Modified human CH4 domain SEQ ID NO: 22 DNA Modified humanCH2-CH4 carrier protein SEQ ID NO: 23 DNA IgE-1 construct SEQ ID NO: 24DNA IgE-2 construct SEQ ID NO: 25 DNA IgE-3 construct SEQ ID NO: 26 DNAIgE-4 construct SEQ ID NO: 27 DNA IgE-5 construct SEQ ID NO: 28 DNA

[0139]

1 28 1 114 PRT Dog CH3 domain 1 Ser Glu Ser Asp Pro Arg Gly Val Thr SerTyr Leu Ser Pro Pro Ser 1 5 10 15 Pro Leu Asp Leu Tyr Val His Lys AlaPro Lys Ile Thr Cys Leu Val 20 25 30 Val Asp Leu Ala Thr Met Glu Gly MetAsn Leu Thr Trp Tyr Arg Glu 35 40 45 Ser Lys Glu Pro Val Asn Pro Gly ProLeu Asn Lys Lys Asp His Phe 50 55 60 Asn Gly Thr Ile Thr Val Thr Ser ThrLeu Pro Val Asn Thr Asn Asp 65 70 75 80 Trp Ile Glu Gly Glu Thr Tyr TyrCys Arg Val Thr His Pro His Leu 85 90 95 Pro Lys Asp Ile Val Arg Ser IleAla Lys Ala Pro Gly Lys Arg Ala 100 105 110 Pro Pro 2 117 PRT HumanCH3/dog CH3 domain fusion 2 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser AlaTyr Leu Ser Arg Pro 1 5 10 15 Ser Pro Phe Asp Leu Phe Ile Arg Lys SerPro Thr Ile Leu Cys Leu 20 25 30 Val Leu Asp Leu Ala Pro Ser Lys Gly ThrVal Gln Leu Thr Trp Ser 35 40 45 Arg Ala Ser Gly Lys Pro Val Asn His SerThr Arg Lys Glu Glu Lys 50 55 60 Asp His Phe Asn Gly Thr Ile Thr Val ThrSer Thr Leu Pro Val Asn 65 70 75 80 Thr Asn Asp Trp Ile Glu Gly Glu ThrTyr Tyr Cys Arg Val Thr His 85 90 95 Pro His Leu Pro Lys Asp Ile Val ArgSer Ile Ala Lys Ala Pro Gly 100 105 110 Lys Arg Ala Pro Pro 115 3 115PRT Human CH3/dog CH3 domain chimera 3 Ala Asp Ser Asn Pro Arg Gly ValThr Ser Tyr Leu Ser Pro Pro Ser 1 5 10 15 Pro Leu Asp Leu Tyr Ile ArgLys Ser Pro Lys Ile Thr Cys Leu Val 20 25 30 Val Asp Leu Ala Pro Ser LysGly Thr Val Asn Leu Thr Trp Ser Arg 35 40 45 Ala Ser Gly Lys Pro Val AsnHis Ser Thr Arg Lys Glu Glu Lys Gln 50 55 60 Arg Asn Gly Thr Ile Thr ValThr Ser Thr Leu Pro Val Gly Thr Arg 65 70 75 80 Asp Trp Ile Glu Gly GluThr Tyr Tyr Cys Arg Val Thr His Pro His 85 90 95 Leu Pro Lys Asp Ile ValArg Ser Ile Ala Lys Ala Pro Gly Lys Arg 100 105 110 Ala Pro Pro 115 4115 PRT Human CH3 4 Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu SerArg Pro Ser 1 5 10 15 Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr IleCys Cys Leu Val 20 25 30 Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn LeuThr Trp Ser Arg 35 40 45 Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg LysGlu Glu Lys Gln 50 55 60 Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu ProVal Gly Thr Arg 65 70 75 80 Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys ArgVal Thr His Pro His 85 90 95 Leu Pro Arg Ala Leu Met Arg Ser Thr Thr LysThr Ser Gly Pro Arg 100 105 110 Ala Ala Pro 115 5 114 PRT Rat CH3 5 SerAsp Asp Glu Pro Arg Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser 1 5 10 15Pro Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys Leu Thr Cys Leu Val 20 25 30Leu Asp Leu Glu Ser Glu Glu Asn Ile Thr Val Thr Trp Val Arg Glu 35 40 45Arg Lys Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser Thr Lys His His 50 55 60Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Asp Ala Lys Asp 65 70 7580 Trp Ile Glu Gly Glu Gly Tyr Gln Cys Arg Val Asp His Pro His Phe 85 9095 Pro Lys Pro Ile Val Arg Ser Ile Thr Lys Ala Pro Gly Lys Arg Ser 100105 110 Ala Pro 6 129 PRT Baculovirus expressed human CH3 domain 6 MetLys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile 1 5 10 15Ser Tyr Ile Tyr Ala Asp Ser Asn Pro Arg Ala Val Ser Ala Tyr Leu 20 25 30Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 35 40 45Leu Cys Leu Val Leu Asp Leu Ala Pro Ser Lys Gly Thr Val Gln Leu 50 55 60Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys 65 70 7580 Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro 85 9095 Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val 100105 110 Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr115 120 125 Ser 7 128 PRT Modified Human CH2 domain 7 Met Ser Val ProThr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala ArgCys Asp Ile Val Ala Ser Arg Asp Phe Thr Pro Pro Ser 20 25 30 Val Lys IleLeu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro 35 40 45 Thr Ile GlnLeu Tyr Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile 50 55 60 Gln Ile ThrTrp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser 65 70 75 80 Thr AlaSer Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu 85 90 95 Leu ThrLeu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Phe Thr Cys 100 105 110 GlnVal Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys 115 120 1258 108 PRT Modified Human CH4 Domain 8 Arg Ala Pro Pro Glu Val Tyr AlaPhe Ala Thr Pro Glu Trp Pro Gly 1 5 10 15 Ser Arg Asp Lys Arg Thr LeuAla Cys Leu Val Gln Asn Phe Met Pro 20 25 30 Glu Asp Ile Ser Val Arg TrpLeu His Asn Glu Val Gln Leu Pro Asp 35 40 45 Ala Arg His Ser Thr Thr GlnPro Arg Lys Thr Lys Gly Ser Gly Phe 50 55 60 Phe Val Phe Ser Arg Leu AlaVal Thr Arg Ala Glu Trp Gln Glu Lys 65 70 75 80 Asp Glu Phe Ile Cys ArgAla Ile His Glu Ala Ala Ser Pro Ser Gln 85 90 95 Thr Val Gln Arg Ala ValSer Val Asn Pro Gly Lys 100 105 9 236 PRT Modified Human CH2-CH4 carrierprotein 9 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp LeuThr 1 5 10 15 Asp Ala Arg Cys Asp Ile Val Ala Ser Arg Asp Phe Thr ProPro Ser 20 25 30 Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His PhePro Pro 35 40 45 Thr Ile Gln Leu Tyr Cys Leu Val Ser Gly Tyr Thr Pro GlyThr Ile 50 55 60 Gln Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val AspLeu Ser 65 70 75 80 Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser ThrGln Ser Glu 85 90 95 Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg ThrPhe Thr Cys 100 105 110 Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp SerThr Lys Lys Cys 115 120 125 Arg Ala Pro Pro Glu Val Tyr Ala Phe Ala ThrPro Glu Trp Pro Gly 130 135 140 Ser Arg Asp Lys Arg Thr Leu Ala Cys LeuVal Gln Asn Phe Met Pro 145 150 155 160 Glu Asp Ile Ser Val Arg Trp LeuHis Asn Glu Val Gln Leu Pro Asp 165 170 175 Ala Arg His Ser Thr Thr GlnPro Arg Lys Thr Lys Gly Ser Gly Phe 180 185 190 Phe Val Phe Ser Arg LeuAla Val Thr Arg Ala Glu Trp Gln Glu Lys 195 200 205 Asp Glu Phe Ile CysArg Ala Ile His Glu Ala Ala Ser Pro Ser Gln 210 215 220 Thr Val Gln ArgAla Val Ser Val Asn Pro Gly Lys 225 230 235 10 346 PRT IgE-1 fusionprotein 10 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp LeuThr 1 5 10 15 Asp Ala Arg Cys Asp Ile Val Ala Ser Arg Asp Phe Thr ProPro Ser 20 25 30 Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His PhePro Pro 35 40 45 Thr Ile Gln Leu Tyr Cys Leu Val Ser Gly Tyr Thr Pro GlyThr Ile 50 55 60 Gln Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val AspLeu Ser 65 70 75 80 Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser ThrGln Ser Glu 85 90 95 Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg ThrPhe Thr Cys 100 105 110 Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp SerThr Lys Lys Cys 115 120 125 Ser Glu Ser Asp Pro Arg Gly Val Thr Ser TyrLeu Ser Pro Pro Ser 130 135 140 Pro Leu Asp Leu Tyr Val His Lys Ala ProLys Ile Thr Cys Leu Val 145 150 155 160 Val Asp Leu Ala Thr Met Glu GlyMet Asn Leu Thr Trp Tyr Arg Glu 165 170 175 Ser Lys Glu Pro Val Asn ProGly Pro Leu Asn Lys Lys Asp His Phe 180 185 190 Asn Gly Thr Ile Thr ValThr Ser Thr Leu Pro Val Asn Thr Asn Asp 195 200 205 Trp Ile Glu Gly GluThr Tyr Tyr Cys Arg Val Thr His Pro His Leu 210 215 220 Pro Lys Asp IleVal Arg Ser Ile Ala Lys Ala Pro Gly Lys Arg Ala 225 230 235 240 Pro ProGlu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg 245 250 255 AspLys Arg Thr Leu Ala Cys Leu Val Gln Asn Phe Met Pro Glu Asp 260 265 270Ile Ser Val Arg Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg 275 280285 His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val 290295 300 Phe Ser Arg Leu Ala Val Thr Arg Ala Glu Trp Gln Glu Lys Asp Glu305 310 315 320 Phe Ile Cys Arg Ala Ile His Glu Ala Ala Ser Pro Ser GlnThr Val 325 330 335 Gln Arg Ala Val Ser Val Asn Pro Gly Lys 340 345 11348 PRT IgE-2 fusion protein 11 Met Ser Val Pro Thr Gln Val Leu Gly LeuLeu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Val Ala SerArg Asp Phe Thr Pro Pro Ser 20 25 30 Val Lys Ile Leu Gln Ser Ser Cys AspGly Gly Gly His Phe Pro Pro 35 40 45 Thr Ile Gln Leu Tyr Cys Leu Val SerGly Tyr Thr Pro Gly Thr Ile 50 55 60 Gln Ile Thr Trp Leu Glu Asp Gly GlnVal Met Asp Val Asp Leu Ser 65 70 75 80 Thr Ala Ser Thr Thr Gln Glu GlyGlu Leu Ala Ser Thr Gln Ser Glu 85 90 95 Leu Thr Leu Ser Gln Lys His TrpLeu Ser Asp Arg Thr Phe Thr Cys 100 105 110 Gln Val Thr Tyr Gln Gly HisThr Phe Glu Asp Ser Thr Lys Lys Cys 115 120 125 Ala Asp Ser Asn Pro ArgGly Val Ser Ala Tyr Leu Ser Arg Pro Ser 130 135 140 Pro Phe Asp Leu PheIle Arg Lys Ser Pro Thr Ile Leu Cys Leu Val 145 150 155 160 Leu Asp LeuAla Pro Ser Lys Gly Thr Val Gln Leu Thr Trp Ser Arg 165 170 175 Ala SerGly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Asp 180 185 190 HisPhe Asn Gly Thr Ile Thr Val Thr Ser Thr Leu Pro Val Asn Thr 195 200 205Asn Asp Trp Ile Glu Gly Glu Thr Tyr Tyr Cys Arg Val Thr His Pro 210 215220 His Leu Pro Lys Asp Ile Val Arg Ser Ile Ala Lys Ala Pro Gly Lys 225230 235 240 Arg Ala Pro Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp ProGly 245 250 255 Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Val Gln Asn PheMet Pro 260 265 270 Glu Asp Ile Ser Val Arg Trp Leu His Asn Glu Val GlnLeu Pro Asp 275 280 285 Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr LysGly Ser Gly Phe 290 295 300 Phe Val Phe Ser Arg Leu Ala Val Thr Arg AlaGlu Trp Gln Glu Lys 305 310 315 320 Asp Glu Phe Ile Cys Arg Ala Ile HisGlu Ala Ala Ser Pro Ser Gln 325 330 335 Thr Val Gln Arg Ala Val Ser ValAsn Pro Gly Lys 340 345 12 347 PRT IgE-3 fusion protein 12 Met Ser ValPro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp AlaArg Cys Asp Ile Val Ala Ser Arg Asp Phe Thr Pro Pro Ser 20 25 30 Val LysIle Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro 35 40 45 Thr IleGln Leu Tyr Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile 50 55 60 Gln IleThr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser 65 70 75 80 ThrAla Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu 85 90 95 LeuThr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Phe Thr Cys 100 105 110Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys 115 120125 Ala Asp Ser Asn Pro Arg Gly Val Thr Ser Tyr Leu Ser Pro Pro Ser 130135 140 Pro Leu Asp Leu Tyr Ile Arg Lys Ser Pro Lys Ile Thr Cys Leu Val145 150 155 160 Val Asp Leu Ala Pro Ser Lys Gly Thr Val Gln Leu Thr TrpSer Arg 165 170 175 Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys GluGlu Lys Gln 180 185 190 Arg Asn Gly Thr Ile Thr Val Thr Ser Thr Leu ProVal Gly Thr Arg 195 200 205 Asp Trp Ile Glu Gly Glu Thr Tyr Tyr Cys ArgVal Thr His Pro His 210 215 220 Leu Pro Lys Asp Ile Val Arg Ser Ile AlaLys Ala Pro Gly Lys Arg 225 230 235 240 Ala Pro Pro Glu Val Tyr Ala PheAla Thr Pro Glu Trp Pro Gly Ser 245 250 255 Arg Asp Lys Arg Thr Leu AlaCys Leu Val Gln Asn Phe Met Pro Glu 260 265 270 Asp Ile Ser Val Arg TrpLeu His Asn Glu Val Gln Leu Pro Asp Ala 275 280 285 Arg His Ser Thr ThrGln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe 290 295 300 Val Phe Ser ArgLeu Ala Val Thr Arg Ala Glu Trp Gln Glu Lys Asp 305 310 315 320 Glu PheIle Cys Arg Ala Ile His Glu Ala Ala Ser Pro Ser Gln Thr 325 330 335 ValGln Arg Ala Val Ser Val Asn Pro Gly Lys 340 345 13 347 PRT IgE-4 fusionprotein 13 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp LeuThr 1 5 10 15 Asp Ala Arg Cys Asp Ile Val Ala Ser Arg Asp Phe Thr ProPro Ser 20 25 30 Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His PhePro Pro 35 40 45 Thr Ile Gln Leu Tyr Cys Leu Val Ser Gly Tyr Thr Pro GlyThr Ile 50 55 60 Gln Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val AspLeu Ser 65 70 75 80 Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser ThrGln Ser Glu 85 90 95 Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg ThrPhe Thr Cys 100 105 110 Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp SerThr Lys Lys Cys 115 120 125 Ala Asp Ser Asn Pro Arg Ala Val Ser Ala TyrLeu Ser Arg Pro Ser 130 135 140 Pro Phe Asp Leu Phe Ile Arg Lys Ser ProThr Ile Leu Cys Leu Val 145 150 155 160 Leu Asp Leu Ala Pro Ser Lys GlyThr Val Gln Leu Thr Trp Ser Arg 165 170 175 Ala Ser Gly Lys Pro Val AsnHis Ser Thr Arg Lys Glu Glu Lys Gln 180 185 190 Arg Asn Gly Thr Leu ThrVal Thr Ser Thr Leu Pro Val Gly Thr Arg 195 200 205 Asp Trp Ile Glu GlyGlu Thr Tyr Gln Cys Arg Val Thr His Pro His 210 215 220 Leu Pro Arg AlaLeu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg 225 230 235 240 Ala AlaPro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser 245 250 255 ArgAsp Lys Arg Thr Leu Ala Cys Leu Val Gln Asn Phe Met Pro Glu 260 265 270Asp Ile Ser Val Arg Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala 275 280285 Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe 290295 300 Val Phe Ser Arg Leu Ala Val Thr Arg Ala Glu Trp Gln Glu Lys Asp305 310 315 320 Glu Phe Ile Cys Arg Ala Ile His Glu Ala Ala Ser Pro SerGln Thr 325 330 335 Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 340 34514 346 PRT IgE-5 fusion protein 14 Met Ser Val Pro Thr Gln Val Leu GlyLeu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Val AlaSer Arg Asp Phe Thr Pro Pro Ser 20 25 30 Val Lys Ile Leu Gln Ser Ser CysAsp Gly Gly Gly His Phe Pro Pro 35 40 45 Thr Ile Gln Leu Tyr Cys Leu ValSer Gly Tyr Thr Pro Gly Thr Ile 50 55 60 Gln Ile Thr Trp Leu Glu Asp GlyGln Val Met Asp Val Asp Leu Ser 65 70 75 80 Thr Ala Ser Thr Thr Gln GluGly Glu Leu Ala Ser Thr Gln Ser Glu 85 90 95 Leu Thr Leu Ser Gln Lys HisTrp Leu Ser Asp Arg Thr Phe Thr Cys 100 105 110 Gln Val Thr Tyr Gln GlyHis Thr Phe Glu Asp Ser Thr Lys Lys Cys 115 120 125 Ser Asp Asp Glu ProArg Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser 130 135 140 Pro Leu Asp LeuTyr Glu Asn Gly Thr Pro Lys Leu Thr Cys Leu Val 145 150 155 160 Leu AspLeu Glu Ser Glu Glu Asn Ile Thr Val Thr Trp Val Arg Glu 165 170 175 ArgLys Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser Thr Lys His His 180 185 190Asn Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Asp Ala Lys Asp 195 200205 Trp Ile Glu Gly Glu Gly Tyr Gln Cys Arg Val Asp His Pro His Phe 210215 220 Pro Lys Pro Ile Val Arg Ser Ile Thr Lys Ala Pro Gly Lys Arg Ser225 230 235 240 Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro GlySer Arg 245 250 255 Asp Lys Arg Thr Leu Ala Cys Leu Val Gln Asn Phe MetPro Glu Asp 260 265 270 Ile Ser Val Arg Trp Leu His Asn Glu Val Gln LeuPro Asp Ala Arg 275 280 285 His Ser Thr Thr Gln Pro Arg Lys Thr Lys GlySer Gly Phe Phe Val 290 295 300 Phe Ser Arg Leu Ala Val Thr Arg Ala GluTrp Gln Glu Lys Asp Glu 305 310 315 320 Phe Ile Cys Arg Ala Ile His GluAla Ala Ser Pro Ser Gln Thr Val 325 330 335 Gln Arg Ala Val Ser Val AsnPro Gly Lys 340 345 15 342 DNA Dog CH3 domain 15 tcagagtccg acccccgaggcgtgacgagc tacctgagcc cacccagccc ccttgacctg 60 tatgtccaca aggcgcccaagatcacctgc ctggtagtgg acctggccac catggaaggc 120 atgaacctga cctggtaccgggagagcaaa gaacccgtga acccgggccc tttgaacaag 180 aaggatcact tcaatgggacgatcacagtc acgtctaccc tgccagtgaa caccaatgac 240 tggatcgagg gcgagacctactattgcagg gtgacccacc cgcacctgcc caaggacatc 300 gtgcgctcca ttgccaaggcccctggcaag cgtgcccccc cg 342 16 355 DNA Human/dog CH3 domain fusion 16gcagattcca acccgagagg ggtgagcgcc tacctaagcc ggcccagccc gttcgacctg 60ttcatccgca agtcgcccac gatcacctgt ctggtggtgg acctggcacc cagcaagggg 120accgtgaacc tgacctggtc ccgggccagt gggaagcctg tgaaccactc caccagaaag 180gaggagaaga aggatcactt caatgggacg atcacagtca cgtctaccct gccagtgaac 240accaatgact ggatcgaggg cgagacctac tattgcaggg tgacccaccc gcacctgccc 300aaggacatcg tgcgctccat tgccaaggcc cctggcaagc gtgccccccc ggaag 355 17 345DNA Human/dog CH3 domain chimera 17 gcagattcca acccgagagg ggtgaccagctacctaagcc cgcccagccc gctggacctg 60 tacatccgca agtcgcccaa gatcacctgtctggtggtgg acctggcacc cagcaagggg 120 accgtgaacc tgacctggtc ccgggccagtgggaagcctg tgaaccactc caccagaaag 180 gaggagaagc aacggaatgg gacgatcacagtcacgtcta ccctgccagt gggcaccaga 240 gactggatcg agggcgagac ctactattgcagggtgaccc acccgcacct gcccaaggac 300 atcgtgcgct ccattgccaa ggcccctggcaagcgtgccc ccccg 345 18 345 DNA Human CH3 domain 18 gcagattccaacccgagagg ggtgagcgcc tacctaagcc ggcccagccc gttcgacctg 60 ttcatccgcaagtcgcccac gatcacctgt ctggtggtgg acctggcacc cagcaagggg 120 accgtgaacctgacctggtc ccgggccagt gggaagcctg tgaaccactc caccagaaag 180 gaggagaagcagcgcaatgg cacgttaacc gtcacgtcca ccctgccggt gggcacccga 240 gactggatcgagggggagac ctaccagtgc agggtgaccc acccccacct gcccagggcc 300 ctcatgcggtccacgaccaa gaccagcggc ccgcgtgctg ccccg 345 19 342 DNA Rat CH3 domain 19tcagatgatg agccccgggg tgtgattacc tacctgatcc cacccagtcc cctcgacctg 60tatgaaaatg ggactcccaa acttacctgt ctggttttgg acctggaaag tgaggagaat 120atcaccgtga cgtgggtccg agagcgtaag aagtctatag gttcggcatc ccagaggagt 180accaagcacc ataatgccac aaccagtatc acctccatct tgccagtgga tgccaaggac 240tggatcgaag gtgaaggcta ccagtgcaga gtggaccacc ctcactttcc caagcccatt 300gtgcgttcca tcaccaaggc cccaggcaag cgctcagccc ca 342 20 327 DNA Human CH3domain (baculovirus expressed) 20 gacagcaacc cgagaggggt gagcgcctacctaagccggc ccagcccgtt cgacctgttc 60 atccgcaagt cgcccacgat cacctgtctggtggtggacc tggcacccag caaggggacc 120 gtgaacctga cctggtcccg ggccagtgggaagcctgtga accactccac cagaaaggag 180 gagaagcagc gcaatggcac gttaaccgtcacgtccaccc tgccggtggg cacccgagac 240 tggatcgagg gggagaccta ccagtgcagggtgacccacc cccacctgcc cagggccctc 300 atgcggtcca cgaccaagac ctcctga 32721 384 DNA Modified human CH2 domain 21 atgagtgtgc ccactcaggt cctggggttgctgctgctgt ggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccgccctccgtga agatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatccagctctact gcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggaggacgggcagg tcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctggcctccacac aaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttcacctgccagg tcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgt 384 22315 DNA Modified human CH4 domain 22 gaagtctatg cgtttgcgac gccggagtggccggggagcc gggacaagcg caccctcgcc 60 tgcctggtgc agaacttcat gcctgaggacatctcggtgc gctggctgca caacgaggtg 120 cagctcccgg acgcccggca cagcacgacgcagccccgca agaccaaggg ctccggcttc 180 ttcgtcttca gccgcctggc ggtgaccagggccgaatggc aggagaaaga tgagttcatc 240 tgccgtgcag tccatgaggc agcgagcccctcacagaccg tccagcgagc ggtgtctgta 300 aatcccggta aatga 315 23 699 DNAModified human CH2-CH4 carrier 23 atgagtgtgc ccactcaggt cctggggttgctgctgctgt ggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccgccctccgtga agatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatccagctctact gcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggaggacgggcagg tcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctggcctccacac aaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttcacctgccagg tcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgtgaagtctatgcgtttg cgacgccgga gtggccgggg 420 agccgggaca agcgcaccct cgcctgcctggtgcagaact tcatgcctga ggacatctcg 480 gtgcgctggc tgcacaacga ggtgcagctcccggacgccc ggcacagcac gacgcagccc 540 cgcaagacca agggctccgg cttcttcgtcttcagccgcc tggcggtgac cagggccgaa 600 tggcaggaga aagatgagtt catctgccgtgcagtccatg aggcagcgag cccctcacag 660 accgtccagc gagcggtgtc tgtaaatcccggtaaatga 699 24 1041 DNA IgE-1 construct 24 atgagtgtgc ccactcaggtcctggggttg ctgctgctgt ggcttacaga tgccagatgt 60 gacatcgtcg cctccagggacttcaccccg ccctccgtga agatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccccccgaccatc cagctctact gcctcgtctc tgggtacacc 180 ccagggacta tccagatcacctggctggag gacgggcagg tcatggacgt ggacttgtcc 240 accgcctcta ccacgcaggagggtgagctg gcctccacac aaagcgagct caccctcagc 300 cagaagcact ggctgtcagaccgcaccttc acctgccagg tcacctatca aggtcacacc 360 tttgaggaca gcaccaagaagtgttcagag tccgaccccc gaggcgtgac gagctacctg 420 agcccaccca gcccccttgacctgtatgtc cacaaggcgc ccaagatcac ctgcctggta 480 gtggacctgg ccaccatggaaggcatgaac ctgacctggt accgggagag caaagaaccc 540 gtgaacccgg gccctttgaacaagaaggat cacttcaatg ggacgatcac agtcacgtct 600 accctgccag tgaacaccaatgactggatc gagggcgaga cctactattg cagggtgacc 660 cacccgcacc tgcccaaggacatcgtgcgc tccattgcca aggcccctgg caagcgtgcc 720 cccccggaag tctatgcgtttgcgacgccg gagtggccgg ggagccggga caagcgcacc 780 ctcgcctgcc tggtgcagaacttcatgcct gaggacatct cggtgcgctg gctgcacaac 840 gaggtgcagc tcccggacgcccggcacagc acgacgcagc cccgcaagac caagggctcc 900 ggcttcttcg tcttcagccgcctggcggtg accagggccg aatggcagga gaaagatgag 960 ttcatctgcc gtgcagtccatgaggcagcg agcccctcac agaccgtcca gcgagcggtg 1020 tctgtaaatc ccggtaaatg a1041 25 1050 DNA IgE-2 construct 25 atgagtgtgc ccactcaggt cctggggttgctgctgctgt ggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccgccctccgtga agatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatccagctctact gcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggaggacgggcagg tcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctggcctccacac aaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttcacctgccagg tcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgtgcagattccaacccga gaggggtgag cgcctaccta 420 agccggccca gcccgttcga cctgttcatccgcaagtcgc ccacgatcac ctgtctggtg 480 gtggacctgg cacccagcaa ggggaccgtgaacctgacct ggtcccgggc cagtgggaag 540 cctgtgaacc actccaccag aaaggaggagaagaaggatc acttcaatgg gacgatcaca 600 gtcacgtcta ccctgccagt gaacaccaatgactggatcg agggcgagac ctactattgc 660 agggtgaccc acccgcacct gcccaaggacatcgtgcgct ccattgccaa ggcccctggc 720 aagcgtgccc ccccggaagt ctatgcgtttgcgacgccgg agtggccggg gagccgggac 780 aagcgcaccc tcgcctgcct ggtgcagaacttcatgcctg aggacatctc ggtgcgctgg 840 ctgcacaacg aggtgcagct cccggacgcccggcacagca cgacgcagcc ccgcaagacc 900 aagggctccg gcttcttcgt cttcagccgcctggcggtga ccagggccga atggcaggag 960 aaagatgagt tcatctgccg tgcagtccatgaggcagcga gcccctcaca gaccgtccag 1020 cgagcggtgt ctgtaaatcc cggtaaatga1050 26 1044 DNA IgE-3 construct 26 atgagtgtgc ccactcaggt cctggggttgctgctgctgt ggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccgccctccgtga agatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatccagctctact gcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggaggacgggcagg tcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctggcctccacac aaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttcacctgccagg tcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgtgcagattccaacccga gaggggtgac cagctaccta 420 agcccgccca gcccgctgga cctgtacatccgcaagtcgc ccaagatcac ctgtctggtg 480 gtggacctgg cacccagcaa ggggaccgtgaacctgacct ggtcccgggc cagtgggaag 540 cctgtgaacc actccaccag aaaggaggagaagcaacgga atgggacgat cacagtcacg 600 tctaccctgc cagtgggcac cagagactggatcgagggcg agacctacta ttgcagggtg 660 acccacccgc acctgcccaa ggacatcgtgcgctccattg ccaaggcccc tggcaagcgt 720 gcccccccgg aagtctatgc gtttgcgacgccggagtggc cggggagccg ggacaagcgc 780 accctcgcct gcctggtgca gaacttcatgcctgaggaca tctcggtgcg ctggctgcac 840 aacgaggtgc agctcccgga cgcccggcacagcacgacgc agccccgcaa gaccaagggc 900 tccggcttct tcgtcttcag ccgcctggcggtgaccaggg ccgaatggca ggagaaagat 960 gagttcatct gccgtgcagt ccatgaggcagcgagcccct cacagaccgt ccagcgagcg 1020 gtgtctgtaa atcccggtaa atga 1044 271044 DNA IgE-4 construct 27 atgagtgtgc ccactcaggt cctggggttg ctgctgctgtggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccg ccctccgtgaagatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatc cagctctactgcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggag gacgggcaggtcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctg gcctccacacaaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttc acctgccaggtcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgtgcagat tccaacccgagaggggtgag cgcctaccta 420 agccggccca gcccgttcga cctgttcatc cgcaagtcgcccacgatcac ctgtctggtg 480 gtggacctgg cacccagcaa ggggaccgtg aacctgacctggtcccgggc cagtgggaag 540 cctgtgaacc actccaccag aaaggaggag aagcagcgcaatggcacgtt aaccgtcacg 600 tccaccctgc cggtgggcac ccgagactgg atcgagggggagacctacca gtgcagggtg 660 acccaccccc acctgcccag ggccctcatg cggtccacgaccaagaccag cggcccgcgt 720 gctgccccgg aagtctatgc gtttgcgacg ccggagtggccggggagccg ggacaagcgc 780 accctcgcct gcctggtgca gaacttcatg cctgaggacatctcggtgcg ctggctgcac 840 aacgaggtgc agctcccgga cgcccggcac agcacgacgcagccccgcaa gaccaagggc 900 tccggcttct tcgtcttcag ccgcctggcg gtgaccagggccgaatggca ggagaaagat 960 gagttcatct gccgtgcagt ccatgaggca gcgagcccctcacagaccgt ccagcgagcg 1020 gtgtctgtaa atcccggtaa atga 1044 28 1041 DNAIgE-5 construct 28 atgagtgtgc ccactcaggt cctggggttg ctgctgctgtggcttacaga tgccagatgt 60 gacatcgtcg cctccaggga cttcaccccg ccctccgtgaagatcttaca gtcgtcctgc 120 gacggcggcg ggcacttccc cccgaccatc cagctctactgcctcgtctc tgggtacacc 180 ccagggacta tccagatcac ctggctggag gacgggcaggtcatggacgt ggacttgtcc 240 accgcctcta ccacgcagga gggtgagctg gcctccacacaaagcgagct caccctcagc 300 cagaagcact ggctgtcaga ccgcaccttc acctgccaggtcacctatca aggtcacacc 360 tttgaggaca gcaccaagaa gtgctcagat gatgagccccggggtgtgat tacctacctg 420 atcccaccca gtcccctcga cctgtatgaa aatgggactcccaaacttac ctgtctggtt 480 ttggacctgg aaagtgagga gaatatcacc gtgacgtgggtccgagagcg taagaagtct 540 ataggttcgg catcccagag gagtaccaag caccataatgccacaaccag tatcacctcc 600 atcttgccag tggatgccaa ggactggatc gaaggtgaaggctaccagtg cagagtggac 660 caccctcact ttcccaagcc cattgtgcgt tccatcaccaaggccccagg caagcgctca 720 gccccagaag tctatgcgtt tgcgacgccg gagtggccggggagccggga caagcgcacc 780 ctcgcctgcc tggtgcagaa cttcatgcct gaggacatctcggtgcgctg gctgcacaac 840 gaggtgcagc tcccggacgc ccggcacagc acgacgcagccccgcaagac caagggctcc 900 ggcttcttcg tcttcagccg cctggcggtg accagggccgaatggcagga gaaagatgag 960 ttcatctgcc gtgcagtcca tgaggcagcg agcccctcacagaccgtcca gcgagcggtg 1020 tctgtaaatc ccggtaaatg a 1041

1. An isolated antigenic peptide comprising: (i) amino acid residues ofa CH3 domain of an IgE molecule from a first species; (ii) amino acidresidues of a CH3 domain of an IgE molecule of a second unrelatedspecies, wherein the amino acid residues of the CH3 domain of the IgEmolecule from the first species are conserved in the CH3 domain of theIgE molecule of the second species, the amino acid residues of the CH3domain of the IgE molecule from the second species are not conserved inthe CH3 domain of the IgE molecule of the first species, and theantigenic peptide induces an anti-IgE immune response that does notcause anaphylaxis when administered to an animal.
 2. The antigenicpeptide of claim 1 comprising at least 10 amino acid residues of the CH3domain of the IgE molecule from either the first or second species. 3.The antigenic peptide of claim 1 comprising at least 10 amino acidresidues of the CH3 domain of the IgE molecule from the first speciesand at least 10 amino acid residues of the CH3 domain of the IgEmolecule from the second species.
 4. The antigenic peptide of claim 1comprising a total of between about 28 and about 31 amino acid residuesof the CH3 domain of the IgE molecule from the first species and the CH3domain of the IgE molecule from the second species.
 5. An isolatedantigenic peptide comprising an amino acid sequence of SEQ ID NO: 2 orSEQ ID NO: 3, that induces an anti-IgE immune response that does notcause anaphylaxis when administered to an animal.
 6. An isolatedantigenic peptide comprising an amino acid sequence of SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, orSEQ ID NO: 14, that induces an anti-IgE immune response that does notcause anaphylaxis when administered to an animal.
 7. An isolatedantigenic fusion protein comprising amino acid residues of a CH3 domainof an IgE molecule from a first species flanked by amino acid residuesof a CH3 domain of an IgE molecule of a second unrelated species,wherein the amino acid residues of the CH3 domain of the IgE moleculefrom the first species are conserved in the CH3 domain of the IgEmolecule of the second species, the amino acid residues of the CH3domain of the IgE molecule from the second species are not conserved inthe CH3 domain of the IgE molecule of the first species, and aheterologous protein carrier, wherein the antigenic fusion proteininduces an anti-IgE immune response that does not cause anaphylaxis whenadministered to an animal.
 8. An isolated antigenic fusion proteincomprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 that induces an anti-IgEimmune response that does not cause anaphylaxis when administered to ananimal.
 9. An isolated peptide comprising an amino acid sequence of SEQID NO: 7, SEQ ID NO: 8, or SEQ ID NO:
 9. 10. The isolated antigenicfusion protein of claim 7 or 8 wherein the heterologous protein carriercomprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQID NO:
 9. 11. An isolated polynucleotide sequence encoding an antigenicpeptide comprising amino acid residues of a CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of a CH3domain of an IgE molecule of a second unrelated species, wherein theamino acid residues of the CH3 domain of the IgE molecule from the firstspecies are conserved in the CH3 domain of the IgE molecule of thesecond species, the amino acid residues of the CH3 domain of the IgEmolecule from the second species are not conserved in the CH3 domain ofthe IgE molecule of the first species, and the antigenic peptide inducesan anti-IgE immune response that does not cause anaphylaxis whenadministered to an animal.
 12. An isolated polynucleotide sequenceencoding an antigenic peptide comprising a nucleic acid sequence of SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, or SEQ ID NO: 28, wherein said antigenic peptide inducesan anti-IgE immune response that does not cause anaphylaxis whenadministered to an animal.
 13. An isolated polynucleotide sequencecomprising a nucleic acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO:
 23. 14. An isolated polynucleotide sequence encoding an antigenicfusion protein comprising amino acid residues of a CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of a CH3domain of an IgE molecule of a second unrelated species, wherein theamino acid residues of the CH3 domain of the IgE molecule from the firstspecies are conserved in the CH3 domain of the IgE molecule of thesecond species, the amino acid residues of the CH3 domain of the IgEmolecule from the second species are not conserved in the CH3 domain ofthe IgE molecule of the first species, and a heterologous proteincarrier, wherein the antigenic fusion protein induces an anti-IgE immuneresponse that does not cause anaphylaxis when administered to an animal.15. An isolated polynucleotide sequence encoding an antigenic fusionprotein comprising a nucleic acid sequence of SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, wherein saidantigenic fusion protein induces an anti-IgE immune response that doesnot cause anaphylaxis when administered to an animal.
 16. An isolatedpolynucleotide sequence encoding an antigenic fusion protein comprisingan amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, wherein theantigenic fusion protein induces an anti-IgE immune response that doesnot cause anaphylaxis when administered to an animal.
 17. A geneticallyengineered host cell that contains the polynucleotide sequence of claim11, 12, 13, or
 14. 18. A genetically engineered host cell that containsthe polynucleotide sequence of claim 11, 12, 13, or 14 in operativeassociation with a nucleotide regulatory sequence that controlsexpression of the polynucleotide sequence in the host cell.
 19. Apharmaceutical composition for inducing an anti-IgE immune response thatdoes not cause anaphylaxis, comprising one or more antigenic peptideshaving an amino acid sequence comprising amino acid residues of a CH3domain of an IgE molecule or a fragment thereof species and apharmaceutically acceptable carrier.
 20. The pharmaceutical compositionof claim 18, wherein at least one antigenic peptide has the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO:
 14. 21. A pharmaceutical composition forinducing an anti-IgE immune response that does not cause anaphylaxiscomprising one or more antigenic fusion proteins having an amino acidsequence comprising amino acid residues of a CH3 domain of an IgEmolecule or a fragment thereof, a heterologous carrier protein and apharmaceutically acceptable carrier.
 22. The pharmaceutical compositionof claim 20, wherein at least one antigenic fusion protein has the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:
 14. 23.The pharmaceutical composition of claim 18 or 20, wherein theheterologous carrier protein is selected from the group consisting ofKLH, PhoE, rmLT, TraT, gD from BHV-1 virus, SEQ ID NO: 7, SEQ ID NO: 8and SEQ ID NO:
 9. 24. The pharmaceutical composition of claim 18 or 20,further comprising an adjuvant.
 25. The pharmaceutical composition ofclaim 18 or 20, wherein the anti-IgE immune response is the productionof anti-IgE antibodies which bind to soluble IgE in serum and otherbodily fluids, prevent IgE from binding to its high affinity receptorson mast cells and basophils, and do not cross-link receptor-bound IgE.26. The pharmaceutical composition of claim 18 or 20 further comprisingan adjuvant.
 27. A pharmaceutical composition for inducing an anti-IgEimmune response that does not cause anaphylaxis comprising one or morepolynucleotide sequences encoding an antigenic peptide having an aminoacid sequence comprising amino acid residues of a CH3 domain of an IgEmolecule or a fragment thereof.
 28. The pharmaceutical composition ofclaim 26, wherein at least one polynucleotide sequence encodes anantigenic peptide having the amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:
 14. 29. Apharmaceutical composition for inducing an anti-IgE immune response thatdoes not cause anaphylaxis comprising one or more polynucleotidesequences encoding an antigenic fusion protein having an amino acidsequence comprising amino acid residues of a CH3 domain of an IgEmolecule or a fragment thereof and a heterologous carrier protein. 30.The pharmaceutical composition of claim 28, wherein at least onepolynucleotide sequence encodes an antigenic fusion protein having theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:
 14. 31. Thepharmaceutical composition of claim 28, wherein the heterologous carrierprotein is selected from the group consisting of KLH, PhoE, rmLT, TraT,gD from BHV-1 virus, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
 9. 32. Amethod for the treating or preventing an IgE-mediated allergic disordercomprising administering to an animal in which such treatment orprevention is desired a immunogenically effective amount of one or moreantigenic peptides having an amino acid sequence comprising amino acidresidues of a CH3 domain of an IgE molecule or a fragment thereof. 33.The method of claim 31, wherein at least one antigenic peptide has theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, orSEQ ID NO:
 14. 34. A method for treating or preventing an IgE-mediatedallergic disorder comprising administering to an animal in which suchtreatment or prevention is desired a immunogenically effective amount ofone or more antigenic fusion protein having an amino acid sequencecomprising amino acid residues of a CH3 domain of an IgE molecule or afragment thereof and a heterologous carrier protein.
 35. The method ofclaim 33, wherein at least one antigenic fusion protein has the aminoacid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO:
 14. 36. A method for the treating or preventing anIgE-mediated allergic disorder comprising administering to an animal inwhich such treatment or prevention is desired a immunogenicallyeffective amount of one or more polynucleotide sequences encoding one ormore antigenic peptides having an amino acid sequence comprising aminoacid residues of a CH3 domain of an IgE molecule or a fragment thereof.37. The method of claim 33, wherein at least one antigenic peptide hasthe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, orSEQ ID NO:
 14. 38. A method for treating or preventing an IgE-mediatedallergic disorder comprising administering to an animal in which suchtreatment or prevention is desired a immunogenically effective amount ofone or more polynucleotide sequences encoding one or more antigenicfusion proteins having an amino acid sequence comprising amino acidresidues of a CH3 domain of an IgE molecule or a fragment thereof and aheterologous carrier protein.
 39. The method of claim 37, wherein atleast one antigenic peptide has the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, or SEQ ID NO:
 14. 40. The method of claim 29, 31, 33 or 35 in whichthe animal is human.
 41. The method of claim 29, 31, 33 or 35 in whichthe animal is a dog.
 42. The method of claim 29, 31, 33 or 35 whereinthe IgE-mediated allergic disorder is asthma, allergic rhinitis,gastrointestinal allergies such as food allergies, eosinophilia,conjunctivitis, or glomerular nephritis, or flea allergies and atopicdermatitis.